M.D. Helmut ; Neumann
email: helmut.neumann@uk-erlangen.de

Florian S Fuchs

Michael Vieth

Raja Atreya

Jürgen Siebler

Ralf Kiesslich

Markus F Neurath

in vivo imaging by endocytoscopy

Keywords: Devices < Topics, Diagnostic tests < Topics, Colonoscopy < Topics, Endoscopy < Topics Endocytoscopy, Ecy, eCLE, pCLE, iCLE, endomicroscopy, red flag technique, in vivo, confocal, magnification, Barrett, IBD, ulcerative colitis, polyp, cancer, celiac disease, Helicobacter pylori, endoscopy, methylene blue, chromoendoscopy, toluidine blue, review, systematic, advanced endoscopic imaging, pEC, iEC esophageal squamous cell cancer,

Introduction: Endocytoscopy (EC) enables in vivo microscopic imaging at 1400-fold magnification, thereby allowing the analysis of mucosal structures at the cellular level. In contrast to fluorescence imaging with confocal laser endomicroscopy which allows analysis of mucosal structures up to 250 µm in depth, EC is based on the principle of contact light microscopy and only allows visualization of the very superficial mucosal layer.

Aim: To systematically review the feasibility and diagnostic yield of EC for in vivo diagnosis of diseases.

Material and Methods:

A systematic search of the literature on diagnostic interventions in the gastrointestinal tract using EC was performed by searches in MEDLINE, Current Contents, PubMed, cross references and references from relevant articles using the search terms "endocytoscopy", "endocytoscope", "magnification endoscopy", "endocytoscopic imaging", "virtual histology", and "optical biopsy". Only full manuscripts and case reports published in English were included.

Results: Overall twenty-nine relevant reports were identified. EC was feasible to detect esophageal squamous cell cancer with sensitivity, specificity and accuracy of 95%, 84% and 82%, respectively. Moreover, EC reached excellent sensitivity and specifity for in vivo diagnosis of colon polyps (91% and 100%, respectively). Other diagnostic applications of EC included diagnosis of Barrett´s oesophagus, Helicobacter pylori, celiac disease, and small cell lung cancer. No serious complications of EC have yet been reported.

Conclusions: EC is a safe and effective new endoscopic imaging technique to obtain in vivo histology and guided biopsies with high diagnostic accuracy. Therefore, EC has the potential to facilitate both diagnosis and patient management.

Recent advances in endoscopic imaging have substantially influenced the diagnostic approach of various gastrointestinal diseases and our understanding of disease pathogenesis. Traditionally, standard white light endoscopy only allows the investigation of mucosal surfaces and surrounding blood vessels at a magnification of about 70-fold. Subsequent histopathological analysis of biopsy specimens remained the gold standard for final diagnosis and corresponding patients management.

Emerging endoscopic imaging modalities including both, vital (e.g. methylene blue, cresyl violet) and virtual chromoendoscopy (Narrow band imaging, NBI; Fujinon intelligent color enhancement, FICE; i-Scan) and magnification endoscopy enabled the endoscopist to visualize and interpret increasing mucosal details [START_REF] Neumann | New endoscopic approaches in IBD[END_REF].

By introducing confocal laser endomicroscopy in 2004 in vivo microscopic imaging at 1000-fold magnification became possible, now allowing the endoscopist to obtain real time in vivo optical biopsies during ongoing endoscopy [START_REF] Kiesslich | Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo[END_REF]. Currently, two endomicroscopy systems are available. One is integrated in the distal tip of a highresolution standard video endoscope (iCLE; Pentax, Tokyo, Japan) one is probebased and capable of passage through the accessory channel of a standard endoscope (pCLE; Cellvizio, Mauna Kea Technologies, Paris, France). Various 1). Nevertheless, to date no systematic review on this emerging endoscopic imaging technique has been performed. The aim of this systematic review was therefore to summarize the diagnostic yield and relevance of endocytoscopy in the clinical setting.

Methods and Techniques

A systematic search of the literature on diagnostic interventions in the gastrointestinal tract using endocytoscopy was performed by searches of MEDLINE, Current Contents, PubMed, cross references and references from relevant articles using the search terms "endocytoscopy", "endocytoscope", "magnification endoscopy", "endocytoscopic imaging", "virtual histology", and "optical biopsy". Only papers and case reports published as full text in English were included. Abstracts were not considered for this review. The literature search was performed in December 2010.

Overall, twenty-nine suitable manuscripts on endocytoscopy were finally identified.

Techniques of endocytoscopy

Endocytoscopy is based on the principle of contact light microscopy. The system consists of a fixed-focus, high-power objective lens that projects magnified images from the sampling site onto a charge-coupled device [4].

Mucosal preparation

To enable ultra high-magnification imaging using the endocytoscope adequate bowel preparation and mucosal preparation is necessary. Based on its principle of contact light microscopy and in contrast to endomicroscopy, endocytoscopy only allows visualization of the very superficial mucosal layer. The light focusing depth ranges from 0 to 50 µm. Therefore, mucolysis using N-acetylcysteine is recommended before the procedure to remove surface mucus [4]. Additionally, prestaining of the mucosa with an absorptive agent is mandatory. One recent study evaluated the optimal staining regimen for endocytoscopy (pEC; XEC-300) using different dye concentrations of crystal violet, methylene blue and toluidine blue [START_REF] Kodashima | Ex-vivo study of high-magnification chromoendoscopy in the gastrointestinal tract to determine the optimal staining conditions for endocytoscopy[END_REF]. Optimal conditions for endocytoscopic mucosal imaging were found after staining with 1% methylene blue in the esophagus and with 0.25% toluidine blue in the stomach and colon. The dyes are usually sprayed onto the mucosal surface using standard spraying catheters. After an appropriate time of exposure (about 60 seconds) to the dye, repeat washing of the mucosa is necessary to remove excess contrast dye before high magnification imaging. While using absorptive contrast agents, for extended visualization repeat staining of the mucosa may be needed [4].

Noteworthily, previous tattooing of suspicious lesions (for example for surveillance examinations) may limit the use of endocytoscopy (pEC; XEC-300) due to decreased image quality because the black pigment is likely to interfere optically with staining of crypts [START_REF] Fasoli | Endocytoscopic imaging and tattooing: a caveat[END_REF]. Currently, two types of endocytoscopes are available (Table 2) [4]. One is integrated in the distal tip of a standard endoscope (iEC) and one is probe-based (pEC, Figure 1). Endocytoscopy images are displayed on a monitor at 30 frames per second, which corresponds to the frame rate during routine high-resolution video endoscopy.

Integrated endocytoscopy systems use two separated lenses and are integrated into an 80-fold magnification endoscope enabling 450-fold magnification (field of view 400

x 400 µm). Recently, another endocytoscopy system was introduced consisting of only one lens that can consecutively increase the magnification up to 380-fold magnification using a hand lever (GIF-Y0002; field of view 700x600 µm). For the first time, this new endoscope-generation enables continues magnification from standard overview to endocytoscopy therefore representing an ´all-in-one´ scope. Upper endoscopes (XGIF-Q260EC1) are 103 cm and lower endoscopes (XCF-Q260EC1) 133 cm in length (Table 2).

Probe-based endocytoscopy consist of handheld miniprobes, which are capable of being inserted through the accessory channel of a standard endoscope. Two different pEC devices exist, providing either 450-fold (XEC 300F) or 1390-fold (XEC 120U) magnification images on a 19-inch monitor. The probes are 380 cm in length and 3.2 mm in diameter thus requiring an accessory channel of 3.7 mm. By introducing the pEC-device through the working channel targeted optical biopsy under guidance of the mother endoscope becomes possible. Additionally, two monitors can be used during the procedure; one is displaying the endoscopic picture, one the in vivo histologic picture. To avoid motion artefacts during the endocytoscopic observation of mucosal structures, a soft short (4 mm) plastic cap is recommended to be affixed at the tip of the endoscope (Figure 2). By using slight suctioning the probe is stabilized against the mucosal wall and high-magnification x 300 µm (0.09 mm 2 ) for the 450-fold magnification probe and with 120 x 120 µm for the 1390-fold magnification probe.

Interpretation

Interpretation of optical biopsies is based on several architectural details (e.g. epithelial structure), cellular features (e.g. size, arrangement of cells) and vascular patterns (e.g. size, leakage, and tortuosity). In addition to these factors, endocytoscopy enables the assessment of cytological features, such as the density of cells, the size and shape of nuclei, and the nucleus-to-cytoplasm ratio (Figure 3) [4]. These are inevitable features for the diagnosis of intraepithelial neoplasia (formerly termed dysplasia) and cancer. Additionally, by appraisal of the staining pattern differentiation of mucosal inflammatory cells become possible (personal unpublished data). Eberl et al. evaluated if endocytoscopy (pEC; XEC-120 and XEC-300) could predict histology in neoplastic lesions [START_REF] Eberl | Can an endocytoscope system (ECS) predict histology in neoplastic lesions?[END_REF]. Therefore, twenty-five patients with esophageal lesions, twenty-eight patients with colonic lesions, and twenty-three patients with gastric lesions were examined. The sensitivity and specificity for the evaluation of the blinded pathologist was 81% and 100%, respectively, in the esophagus, 56% and 89% in the stomach, and 79% and 90% in the colon.

Nevertheless, to date no prospective study has validated endocytoscopy-based image criteria. Importantly, if an endoscopist evaluated the endocytoscopic pictures in combination with the macroscopic endoscopic images sensitivity and specificity increased significantly (sensitivity 96%, specificity 95%). As mentioned above, endocytoscopy is based on the principle of contact light microscopy. In vivo imaging is achieved by a fixed-focus, high-power objective lens that projects images onto a charge-coupled device [4]. Therefore, the depth of field is dependent on the absorption spectra and light penetration depth of the intestinal tissue and can not be altered manually. Because absorption spectra and light penetration depth are themselves dependent on tissue structure (e.g. squamous epithelium, cylindric epithelium) and composition (e.g. inflammation, haemoglobin) the light focusing depth ranges theoretically from 0 µm up to 50 µm [START_REF] Barun | Absorption spectra and light penetration depth of normal and pathologically altered human skin[END_REF]. Of note it is currently not possible to determine the exact level of endocytoscopic imaging. Furthermore, in order to realize ultra high-magnification imaging up to 1400-fold, prestaining of the mucosa with either methylene blue (Akorn, Lake Forest, USA) or toluidine blue (Dr. Franz Köhler Chemie, Bensheim, Germany) is strictly necessary.

Consequently, endocytoscopy is able to identify cellular (e.g. visibility of nuclei) and architectural features of the surface epithelial layer. In contrast to endomicroscopy, endocytoscopy does not allow the visualization of deeper layers of the epithelium and is therefore not suitable to evaluate the depth of invasion in early neoplasia. In addition, concerns about the use of methylene blue have risen, which may increase DNA damage in the tissue after illumination [START_REF] Hardie | Chromoendoscopy with methylene blue and the risk of DNA damage[END_REF]. As a consequence, methylene blue was recently withdrawn from the German market but is still available in other countries (i.e. USA). Toluidine blue is approved by the Federal Institute for Drugs and Medical Devices for vital staining. To the best of our knowledge, no concerns regarding the safety of topically applied toluidine blue are known. In contrast to endocytoscopy, magnification (also called "zoom") endoscopy uses a movable lens controlled by the endoscopist to vary degree of magnification, ranging from 1.5-fold to 150-fold. Both, high-resolution and magnification features are integrated in one endoscope and enable the endoscopist to analyze the pit pattern (e.g. villiform or distorted in Barrett´s esophagus) of lesions [START_REF] Sharma | Magnification endoscopy[END_REF]. Besides optical magnification endoscopy, modern video processors enable digital magnification features. These are based on special software algorithms which enlarges the original image. Because the resolution of the digital sensor is fixed the software tries to interpolate additional pixels which markedly reduce the quality of the resulting image.

In contrast to endocytoscopy, confocal laser endomicroscopy is based on tissue illumination after application of fluorescence agents which can either be applied systemically (fluorescein) or topically (e.g. acriflavine, cresyl violet). Laser light is focused to a single point within a defined microscopic field of view. The laser light stimulates the fluorescence agent and light emanating from this point is focused through a pinhole to a detector while light emanating from outside the illuminated spot is excluded from detection. The gray scale image created is an optical section representing one focal plane within the specimen [START_REF] Kiesslich | Advanced imaging in endoscopy[END_REF]. The iCLE system offers the operator to manually adjust the imaging plane depth ranging from 0-250 µm using an optical slice thickness of 7 µm. Endomicroscopy offers in vivo imaging of cellular and subcellular structures at 1000-fold magnification compared to 1400-fold magnification of endocytoscopy [START_REF] Neumann | Confocal laser endomicroscopy: technical advances and clinical applications[END_REF]. While concerns about the use of acriflavine have raised (potential mutagenic effects) one recent large multicenter study have addressed the safety of intravenous fluorescein for endomicroscopy in the gastrointestinal tract [START_REF] Wallace | The safety of intravenous fluorescein for confocal laser endomicroscopy in the gastrointestinal tract[END_REF].

Current applications of endocytoscopy

Esophagus

Endoscopic imaging of esophageal disorders is well established in clinical practice due to the increasing incidence of cancers at this location and their poor prognosis in advanced disease stages. Moreover, the opportunity of gentle curative endoscopic resection methods in early disease stages increases the need for imaging modalities which allow the detection of subtle mucosal changes with a high diagnostic yield (Table 3).

In 2004, Kumagai et al. observed the surface of the squamous epithelium in normal esophageal mucosa and superficial esophageal cancers using endocytoscopy (pEC; XEC-120 and XEC-300) [START_REF] Kumagai | Magnifying chromoendoscopy of the esophagus: in-vivo pathological diagnosis using an endocytoscopy system[END_REF]. Normal cells were homogeneously arranged, and the nucleus to cytoplasm ratio was uniform and low. In contrast, in esophageal cancers, the cell distribution and nucleus-to-cytoplasm ratio was irregular and the density of cells was increased. However, no statistic analysis was done in this study.

Additionally, three case reports confirmed the potential of endocytoscopy for in vivo diagnosis of early esophageal squamous cell carcinoma [START_REF] Dumonceau | In vivo microscopic examination of the esophagus for the detection of cancer before liver transplantation in patients with alcoholic cirrhosis[END_REF][START_REF] Fujishiro | Detailed comparison between endocytoscopy and horizontal histology of an esophageal intraepithelial squamous cell carcinoma[END_REF][START_REF] Banerjee | Application of highresolution narrow band imaging and endocytoscopy for early diagnosis of esophageal neoplasia[END_REF]. Using pEC with 450-fold magnification (pEC; XEC-300), it was possible to visualize typical neoplastic mucosal alterations like irregular and enlarged nuclei during ongoing endoscopy.

Inoue et al. conducted a pilot trial including twenty-nine consecutive patients with esophageal lesions who were evaluated using endocytoscopy (iEC; XGIF-Q260EC1) after prestaining of the esophageal mucosa with 0.5% methylene blue [START_REF] Inoue | Endoscopic in vivo evaluation of tissue atypia in the esophagus using a newly designed integrated endocytoscope: a pilot trial[END_REF]. The positive predictive value for malignancy was 94%, the sensitivity 83.3% and specificity 93.7% with an overall accuracy for differentiating between malignant and non-malignant tissue of 82%.

To investigate whether endocytoscopic images of cancerous and non-cancerous esophageal mucosa correspond with conventional histopathology, a multicenter ex 12 vivo pilot study was conducted [START_REF] Fujishiro | Potential and present limitation of endocytoscopy in the diagnosis of esophageal squamous-cell carcinoma: a multicenter ex vivo pilot study[END_REF]. Overall, twenty-seven patients with esophageal squamous cell cancers were included and evaluable pairs of an endocytoscopic image and a histological picture of the same site were obtained at twelve cancerous and fourteen normal areas. The pattern of the cellular arrangement and density and the size and shape of nuclei were morphologically identical between endocytoscopy (pEC; XEC-300) and conventional histopathology. However, the cytoplasm was not identifiable in endocytoscopic images. Mean (± SD) total numbers of nuclei per endocytoscopic image were 129 ± 14.8 at normal areas and 550 ± 66.5 at cancerous areas (P < .0001).

One recent published study aimed at evaluating whether endocytoscopic observation of esophageal squamous cell carcinoma could replace histopathological examination of biopsy specimen [START_REF] Kumagai | Endocytoscopic observation for esophageal squamous cell carcinoma: can biopsy histology be omitted?[END_REF]. Therefore, fifty-seven iodine-unstained areas in resected specimens of the esophagus from twenty-eight patients were examined. Additionally, seventy-one lesions of esophageal squamous cell carcinoma were analyzed in vivo using endocytoscopy (pEC; XEC-120 and XEC-300). The sensitivity and specificity of endocytoscopy for malignant lesions was 94.7% and 84.2%, respectively. In 93.5% of cancerous lesions diagnosed using endocytoscopy further biopsy examinations were considered as unnecessary by the histopathologist.

Barrett´s esophagus is strongly associated with gastroesophageal reflux disease and well recognized as a precursor lesion of esophageal adenocarcinoma. Early detection of mucosal alterations is important to provide an efficient surveillance and endoscopic therapy in those patients.

One study conducted by Pohl and coworkers evaluated the feasibility of endocytoscopy (pEC; XEC-120 and XEC-300) in the surveillance of patients with Barrett´s esophagus [START_REF] Pohl | Evaluation of endocytoscopy in the surveillance of patients with Barrett's esophagus[END_REF]. Overall 166 biopsy sites from sixteen patients without F o r P e e r R e v i e w 13 visible lesions, who presented for Barrett surveillance were analyzed using endocytoscopy and correlated to histopathology. Adequate assessment of endocytoscopy images was possible in 51% with magnification x 450 and in 22% with magnification x 1125. At most, 23% of images with lower magnification were interpretable to identify characteristics of neoplasia, and 41% with higher magnification. Interobserver agreement was fair with kappa values from < 0 to 0.45.

Positive and negative predictive values for high-grade intraepithelial neoplasia or cancer were 0.29 and 0.87, respectively, for magnification x 450 and 0.44 and 0.83, respectively, for magnification x 1125.

Key message:

Endocytoscopy could detect esophageal squamous cell carcinoma with good sensitivity and specificity.

In Barrett´s esophagus endocytoscopy lacks sufficient image quality to be currently of assistance in identifying neoplastic areas.

Stomach:

Helicobacter pylori (H. pylori) infection affects about fifty percent of the general population (10-30% in industralized countries and 80-90% in developing countries) and is strongly associated with gastroduodenal ulcer disease, gastric lymphoma and non-proximal gastric adenocarcinoma. In addition, Fasoli and coworkers published one case report highlighting the potential of endocytoscopy (XEC-300) for in vivo evaluation of gastric signet ring cell carcinoma. Endocytoscopy was reliable to identify disease specific histologic aspects and there was a strong concordance between in vivo and ex vivo histology [START_REF] Fasoli | Signet ring cell carcinoma of the stomach: correlation between endocytoscopy and histology[END_REF].

Key message:

Currently, no studies are available concerning endocytoscopy based in vivo diagnosis of H. pylori infection, hyperplastic and precancerous lesions of the stomach.

Duodenum:

Celiac disease represents an autoimmune enteropathy in genetically susceptible hosts induced by gluten. Histopathological diagnosis is based on villous atrophy, hyperplasia of intestinal crypts, and an increased number of intraepithelial lymphocytes.

Up to now, two studies have evaluated the potential of endocytoscopy for in vivo diagnosis of celiac disease.

In the first study, a total of 166 endocytoscopy (pEC; XEC-120 and XEC-300) recordings from forty patients with established (n= 32) or suspected celiac disease were prospectively acquired and corresponded to conventional histopathology [START_REF] Pohl | Endocytoscopy for the detection of microstructural features in adult patients with celiac sprue: a prospective, blinded endocytoscopy-conventional histology correlation study[END_REF].

When adjusted by multivariate analysis four criteria were identified to be significant predictors for Marsh III pathology: low number of villi per visual field (<3; odds ratio irregular epithelial lining (OR 10.9; 95% CI, 2.5-46.7), and inability to delineate loop capillaries (OR 14.9; 95% CI, 3.3-67.0). Nevertheless, none of these factors was a good predictor for Marsh I pathology.

Sixteen patients with documented celiac disease and seven control patients were included in the second trial, evaluating the potential of endocytoscopy (pEC; XEC-300) in celiac disease [START_REF] Matysiak-Budnik | In vivo real-time imaging of human duodenal mucosal structures in celiac disease using endocytoscopy[END_REF]. Endocytoscopy visualized the presence of normalappearing, long, thin villi, lined with clearly distinguishable surface epithelial cells, the presence of thick, shortened villi, reflecting partial villous atrophy, and the total absence of villi and the presence of enlarged crypt orifices, reflecting total villous atrophy. Sensitivity and specificity of endocytoscopy for the diagnosis of villous atrophy was 88% and 100%, respectively.

Key message:

In celiac disease endocytoscopy enables accurate determination of severe abnormalities like villous atrophy. Nevertheless, mild changes cannot be assessed.

Therefore, diagnosis and follow-up of patients should still be based on biopsies.

Colon:

According to most national and international guidelines, colonoscopy is the recommended surveillance method of patients aged 50 years and older to exclude adenomatous polyps. However, most polyps are diminutive in size (1-5 mm) and harbour only a very low prevalence of cancer [START_REF] Rex | Preventing colorectal cancer and cancer mortality with colonoscopy: what we know and what we don't know[END_REF]. Therefore, sending diminutive polyps to the histopathologist is generally a profusion of resources. By using real time in vivo histology, suspicious polyps could be identified for targeted biopsy or endoscopic polypectomy (Table 4). [START_REF] Sasajima | Real-time in vivo virtual histology of colorectal lesions when using the endocytoscopy system[END_REF]. Endocytoscopy was able to differentiate normal colonic mucosa, hyperplastic polyps, low-grade and high-grade adenomas and invasive cancers. The overall accuracy of endocytoscopy was 93.3% with an excellent kappa value between endocytoscopy and the corresponding histopathological analysis of 0.910. The differential diagnosis between neoplastic and non-neoplastic lesions and between adenoma and invasive cancer was statistically significant (P <.001).

In addition to this context, one recent published case report highlighted the potential of endocytoscopy (pEC; XEC-120) for in vivo diagnosis of very focal high-grade intraepithelial neoplasia in colonic polyps [START_REF] Neumann | Endocytoscopy-based detection of focal highgrade intraepithelial neoplasia in colonic polyps (with video)[END_REF]. This is of tremendous importance, because polyps often show only focal lesions which are surrounded by normal colonic mucosa.

Meroni et al. addressed this point in another case report [START_REF] Meroni | Detection of tissue abnormalities in normal mucosa surrounding colorectal cancer using an endocytoscopy system[END_REF]. They performed endocytoscopy (pEC; XEC-300) in one surgical specimen obtained after resection of colon cancer and found focal abnormalities of colonic glands highly suggestive of aberrant crypt foci seven cm apart from the primary tumor within macroscopically normal mucosa.

Finally, Cipolletta et al. conducted a prospective study including forty-one consecutive patients with colorectal aberrant crypt foci [START_REF] Cipolletta | Endocytoscopy can identify dysplasia in aberrant crypt foci of the colorectum: a prospective in vivo study[END_REF]. The mean duration of the endocytoscopy procedure was 44 ± 12 minutes (range 31 -62 minutes) and it was possible to observe lesions at the cellular level and evaluate both cellular and structural atypia in vivo. In normal mucosa, crypts had preserved individuality and round-shaped contours. Nuclei were located at the basal third of the crypt in a single line, and the lumen was circular. In dysplastic aberrant crypt foci, crypt contours were polygonal, cell nuclei were elongated with pseudostratification toward the luminal half of the crypt and irregularly arranged, and the lumen was linear. Sensitivity and specificity of endocytoscopy (pEC; XEC-300) for the detection of intraepithelial neoplasia was 91.4% and 100%, respectively. Interobserver agreement between endoscopist and pathologist was good (weighted kappa 0.68; 95 % CI 0.59 -0.78).

The same group aimed to access the capability of endocytoscopy (pEC; XEC-300) in differentiating neoplastic from non-neoplastic lesions in the colorectum [START_REF] Rotondano | Endocytoscopic classification of preneoplastic lesions in the colorectum[END_REF]. Overall, fifty-two lesions were examined in 49 patients (17 polypoid and 35 non-polypoid).

Final pathological diagnosis was normal mucosa or hyperplastic polyp in ten cases, low-grade adenoma in twenty-nine, high-grade adenoma in eleven, and submucosal invasive cancer in two cases. Positive predictive value of endocytoscopy was 100% for hyperplastic polyps, 93.1% for low-grade intraepithelial neoplasia, 90.1% for highgrade intraepithelial neoplasia, and 100% for invasive cancer. Additionally, endocytoscopy diagnosis correlated completely with histopathology in the differentiation between neoplastic and non-neoplastic lesions.

One recent case report identified endocytoscopy (pEC; XEC-300) as a potential method for imaging of a carcinoid tumor which was located in the terminal ileum.

Nests of monomorphous cells separated by poor cellulated strands without glandular structures were evident on endocytoscopic imaging [START_REF] Fasoli | Endocytoscopic imaging of a carcinoid tumor[END_REF].

Very recently, Hosoe and coworkers prospectively evaluated the feasibility of endocytoscopy with 450-fold magnification (iEC; CF-Y0001) for the in vivo diagnosis of amebic parasites in five patients. The authors could successfully visualize amebic trophozoites in all cases and therefore diagnose amoebic colitis in vivo during ongoing endoscopy [START_REF] Hosoe | In vivo visualization of trophozoites in patients with amoebic colitis by using a newly developed endocytoscope[END_REF].

Lung:

Very recently, our group addressed the potential of endocytoscopy for in vivo diagnosis of small cell lung cancer during ongoing bronchoscopy in four patients [START_REF] Neumann | In vivo diagnosis of small cell lung cancer by endocytoscopy[END_REF].

After topical application of methylene blue, endocytoscopy (pEC; XEC-120) was able to reliable identify numerous small blue cells with hyperchromatic nuclei (Figure 4).

Corresponding histopathological analysis confirmed in vivo diagnosis of small cell lung cancer. This exciting initial experience is now evaluated in a larger ongoing prospective study.

Shibuya and coworkers performed endocytoscopy (pEC; XEC-300) in twenty-two patients (7 squamous cell carcinoma, 11 squamous dysplasia and 4 after PDT therapies). Both abnormal areas of interest and normal bronchial mucosa were stained with 0.5% methylene blue and afterwards examined with endocytoscopy at 570-fold magnification. In this study, endocytoscopy was useful to discriminate between normal bronchial epithelial cells, dysplastic cells and malignant cells during ongoing bronchoscopy [START_REF] Shibuya | In vivo microscopic imaging of the bronchial mucosa using an endo-cytoscopy system[END_REF]. Besides the above mentioned areas, endocytoscopy was also proven to be efficient in different disease entities.

In a pilot study including five patients, endocytoscopy was used to evaluate bladder carcinoma [START_REF] Ohigashi | Endocytoscopy: novel endoscopic imaging technology for in-situ observation of bladder cancer cells[END_REF]. Using the pEC system with 450-fold magnification (XEC-300), the cell structure and nuclear morphology of the tumors were identified and graded correctly in 80% of the cases.

In another case report endocytoscopy (not nearly specified which system was used) was used to assist intraoperative diagnosis of carcinoma in a patient who was suffering from chronic pancreatitis. Diagnosis of cancer was based on the visualization of cells with hyperchromatic nuclei and a high nucleus to cytoplasm ratio [START_REF] Rao | Endocytoscopy assists in the intraoperative diagnosis of carcinoma in a patient with chronic pancreatitis[END_REF].

While it is often challenging to optimally sample the muscularis propria endoscopically for the diagnosis of muscle layer diseases, especially for motility disorders resulting from neuroenteric dysfunction, Sumiyama and coworkers designed an ex vivo and in vivo porcine animal study to access the muscular layer using endocytoscopy (pEC; XEC-120 and XEC-300) [START_REF] Sumiyama | Pilot study for in vivo cellular imaging of the muscularis propria and ex vivo molecular imaging of myenteric neurons (with video)[END_REF]. Cellular microstructures resembling spindle-shaped smooth muscle cells and neuron-like cells were visualized by endocytoscopy in vivo.

Endocytoscopy was used in another report of two patients for intraoperative diagnosis of disseminated malignancy. During diagnostic laparoscopy, endocytoscopy (pEC; XEC-300) of peritoneal nodules revealed malignant cells and in vivo diagnosis was confirmed by corresponding histopathology [START_REF] Rao | Endocytoscopy assisted laparoscopic intraoperative diagnosis of disseminated malignancy[END_REF].

Additionally, after methylene blue staining onto normal rectal mucosa, endocytoscopy (pEC; XEC-300) revealed red blood cell flow within the microvasculature circulating through the arterioles [START_REF] Yan | In vivo real-time endocytoscopic visualization of blood flow in rectal microvasculature[END_REF]. Therefore, endocytoscopy is a promising new development providing advanced endoscopic imaging. Nevertheless, multicenter studies evaluating the technique in larger settings are highly warranted.

In the near future, new ´all-in-one´ scopes for stable ultra-high magnification imaging and functional imaging could further improve the diagnostic applicability of endocytoscopy.

Statement of interest:

There was no financial support for this study. Table 1: Current potential clinical applications of endocytoscopy.

Esophagus

-Esophageal squamous cell carcinoma [START_REF] Barun | Absorption spectra and light penetration depth of normal and pathologically altered human skin[END_REF][START_REF] Hardie | Chromoendoscopy with methylene blue and the risk of DNA damage[END_REF][START_REF] Sharma | Magnification endoscopy[END_REF][START_REF] Kiesslich | Advanced imaging in endoscopy[END_REF][START_REF] Wallace | The safety of intravenous fluorescein for confocal laser endomicroscopy in the gastrointestinal tract[END_REF][START_REF] Kumagai | Magnifying chromoendoscopy of the esophagus: in-vivo pathological diagnosis using an endocytoscopy system[END_REF][START_REF] Dumonceau | In vivo microscopic examination of the esophagus for the detection of cancer before liver transplantation in patients with alcoholic cirrhosis[END_REF] -Barrett´s esophagus [START_REF] Fujishiro | Detailed comparison between endocytoscopy and horizontal histology of an esophageal intraepithelial squamous cell carcinoma[END_REF] Stomach -Helicobacter pylori -infection [START_REF] Banerjee | Application of highresolution narrow band imaging and endocytoscopy for early diagnosis of esophageal neoplasia[END_REF] -Signet ring cell carcinoma [START_REF] Inoue | Endoscopic in vivo evaluation of tissue atypia in the esophagus using a newly designed integrated endocytoscope: a pilot trial[END_REF] Duodenum -Celiac disease [START_REF] Fujishiro | Potential and present limitation of endocytoscopy in the diagnosis of esophageal squamous-cell carcinoma: a multicenter ex vivo pilot study[END_REF][START_REF] Kumagai | Endocytoscopic observation for esophageal squamous cell carcinoma: can biopsy histology be omitted?[END_REF]

Colon

-Polyps [START_REF] Kimura | Ex vivo visualization of Helicobacter pylori using an endocytoscopic probe[END_REF][START_REF] Fasoli | Signet ring cell carcinoma of the stomach: correlation between endocytoscopy and histology[END_REF][START_REF] Pohl | Endocytoscopy for the detection of microstructural features in adult patients with celiac sprue: a prospective, blinded endocytoscopy-conventional histology correlation study[END_REF][START_REF] Matysiak-Budnik | In vivo real-time imaging of human duodenal mucosal structures in celiac disease using endocytoscopy[END_REF][START_REF] Rex | Preventing colorectal cancer and cancer mortality with colonoscopy: what we know and what we don't know[END_REF][START_REF] Sasajima | Real-time in vivo virtual histology of colorectal lesions when using the endocytoscopy system[END_REF] -Amoebic colitis [START_REF] Neumann | Endocytoscopy-based detection of focal highgrade intraepithelial neoplasia in colonic polyps (with video)[END_REF] Lung -small cell lung cancer [START_REF] Meroni | Detection of tissue abnormalities in normal mucosa surrounding colorectal cancer using an endocytoscopy system[END_REF][START_REF] Cipolletta | Endocytoscopy can identify dysplasia in aberrant crypt foci of the colorectum: a prospective in vivo study[END_REF] Others -Bladder carcinoma [START_REF] Cipolletta | Endocytoscopy can identify dysplasia in aberrant crypt foci of the colorectum: a prospective in vivo study[END_REF] -Intraoperative carcinoma diagnosis [START_REF] Rotondano | Endocytoscopic classification of preneoplastic lesions in the colorectum[END_REF][START_REF] Hosoe | In vivo visualization of trophozoites in patients with amoebic colitis by using a newly developed endocytoscope[END_REF]

F o r P e e r R e v i e w

27 Deleted: E

"

  Whatever you can do or dream you can, begin it. Boldness has genius, power, and magic in it." Johann Wolfgang von Goethe, German poet.

  studies have shown the potential of both types of endomicroscopy-systems for in vivo diagnosis of different disease entities including esophageal squamous cell esophagus, Helicobacter pylori infection, celiac disease, ulcerative colitis and colon polyps [3]. Endocytoscopy (Olympus, Tokyo, Japan) was recently introduced and represents another emerging endoscopic imaging technique enabling real time in vivo diagnosis of cellular patterns at extremely high magnification. Recent published data indicate that endocytoscopy has the potential to change the current paradigm for the diagnosis of various diseases (Table

Formatted:

  Font: Italic Deleted: Besides this fascinating technique, endocytoscopy (Olympus, Tokyo, Japan) allows real time in vivo diagnosis Deleted: likewise. ¶

  become possible. The horizontal observation field is given with 300

9

 9 Comparison of endocytoscopy to magnification endoscopy and endomicroscopyEndocytoscopy, magnification endoscopy and endomicroscopy represent completely different endoscopic techniques and all require totally different endoscopes.

  Kimura et al. cultured H. pylori ex vivo from gastric mucus of three patients sufferingfrom gastric ulcer disease. Subsequently, H. pylori microorganisms could be supernatant of the culture medium using endocytoscopy with 1100fold magnification (pEC; XEC-300)[START_REF] Kimura | Ex vivo visualization of Helicobacter pylori using an endocytoscopic probe[END_REF].

[OR] 9 . 1 ;

 91 95% CI, 1.3-62.0), confluence of villi (OR 37.1; 95% CI, 1.3-1021.2),

  et al. evaluated in a prospective study the usefulness of endocytoscopy (pEC; XEC-120 and XEC-300) for the differentiation of colorectal lesions in 113 consecutive patients

  Endocytoscopy can predict the presence of preneoplastic and neoplastic lesions in the colon with high accuracy. One drawback is the prolongation up to one hour of the duration of endoscopy.

21 instruments

 21 Potential applications of endocytoscopy were shown in different pilot studies including lung, bladder and intraoperative diagnosis of malignancy. Larger trials are now needed to confirm these interesting and promising preliminary results.ConclusionsModern endoscopy with advanced endoscopic imaging techniques has revolutionized the diagnosis and management of patients in the endoscopy suite. Besides other emerging endoscopic imaging techniques, including chromoendoscopy, magnification endoscopy and confocal laser endomicroscopy, endocytoscopy has become an inherent part of advanced endoscopic imaging. In different disease entities, endocytoscopy allowed the endoscopist to obtain real time in vivo histology during ongoing endoscopy with a high diagnostic yield and to guide subsequent biopsy acquisition for histopathological analysis. Up to now, no serious complications of endocytoscopy have been reported. Currently, the spectrum of endocytoscopy ranges from esophageal disorders, including esophageal squamous cell carcinoma and Barrett´s esophagus to celiac disease and in vivo diagnosis of colon polyps and intestinal pathogens like Helicobacter pylori and amebic trophozoites. Besides, the value of endocytoscopy was also proven for the in vivo diagnosis of small cell lung cancer and to facilitate final histological diagnosis during diagnostic laparoscopy.Nevertheless, most studies so far only include small sample sizes or are case reports. No multicenter trial evaluating the technique has yet been performed. In addition, as endocytoscopy is a developing technology the performance of different varies widely. Up to date, no information regarding the learning curve or the inter-observer and intra-observer agreement of endocytoscopy are available.

Figure 1 :

 1 Figures:

29 Figure 2 :

 292 Figure 2: After mucosal staining with methylene blue the probe-based endocytoscope is gently pushed against the mucosal surface to enable highmagnification imaging. Note the plastic cap at the distal end of the endoscope to reduce motion artefacts.

30 Figure 3 :

 303 Figure 3: Optical biopsy at 450-fold (Panel A) and 1390-fold (Panel B) magnification using the endocytoscope highlights density of cells, the size and shape of nuclei, and the nucleus-to-cytoplasm ratio. Panel A shows normal gastric epithelium after toluidine blue staining. Microvessels are clearly visible (arrows). Panel B highlights normal colonic epithelium after toluidine blue staining.

Figure 4 :

 4 Figure 4: Methylene-blue aided endocytoscopy in a patient with small cell lung cancer visualizes numerous small blue cells with hyperchromatic nuclei.

Table 2 : Technical characteristics of different endocytoscopy devices.

 2

	Type	Probe-based (pEC)	Endoscope-based (iEC)
		XEC-300	XEC-120	XGIF-	XCF-	GIF-Y0002
				Q260E	Q260E
				C1	C1
	Endoscope		N/A	x 85	x 110	x 380
	magnification					(x 600 digital)
	Endoscope working		N/A	2.8	3.2	2.8
	channel (mm)
	Total length (cm)		380	103	133	103
	Functional length (cm)		240	103	133	103
	EC magnification	x 450 (14´ monitor)	x 1100 (14´ monitor)	x 580	x 380
		x 570 (19´monitor)	x 1390 (19´ monitor)			(x 600 digital)
	Field of view (µm)	300 x 300	120 x 120	400 x 400	700 x 600
						(440 x 380 digital)
	Horizontal resolution	4.2	1.7		4.0	3.7
	Outer diameter		3.2 mm	11.6	13.6	10.7
	Imaging plane depth	30 µm		5 µm		50 µm
	EC =endocytoscope.

Table 3 : Summary of studies evaluating endocytoscopy for diagnosing esophageal diseases.

 3

Alimentary Pharmacology & Therapeutic

Author and reference Disease entity

Study design Patients Results [START_REF] Kumagai | Magnifying chromoendoscopy of the esophagus: in-vivo pathological diagnosis using an endocytoscopy system[END_REF] [START_REF] Barun | Absorption spectra and light penetration depth of normal and pathologically altered human skin[END_REF] SCC Descriptive study 12 Dumonceau JM, 2006 [START_REF] Hardie | Chromoendoscopy with methylene blue and the risk of DNA damage[END_REF] SCC Case report 1 Fujishiro M, 2008 [START_REF] Sharma | Magnification endoscopy[END_REF] SCC Case report 1 Banerjee R, 2008 [START_REF] Kiesslich | Advanced imaging in endoscopy[END_REF] SCC Case series 3

In cancerous mucosa, cell distribution and nucleus-to-cytoplasm ratio was irregular and density of cells increased. [START_REF] Kimura | Ex vivo visualization of Helicobacter pylori using an endocytoscopic probe[END_REF] Prospective study 113 Accuracy 93.3%; kappa between EC and histopathology 0.91 Neumann H, 2010 [START_REF] Fasoli | Signet ring cell carcinoma of the stomach: correlation between endocytoscopy and histology[END_REF] Case report 1 EC could detect focal HGIEN Meroni E, 2007 [START_REF] Pohl | Endocytoscopy for the detection of microstructural features in adult patients with celiac sprue: a prospective, blinded endocytoscopy-conventional histology correlation study[END_REF] Case report 1 EC could detect tissue abnormalities in normal mucosa surrounding colorectal cancer Cipolletta L, 2009 [START_REF] Matysiak-Budnik | In vivo real-time imaging of human duodenal mucosal structures in celiac disease using endocytoscopy[END_REF] Prospective study 41 Sensitivity 91.4%, Specificity 100%, Interobserver agreement 0.68 Rotondano G, 2010 [START_REF] Rex | Preventing colorectal cancer and cancer mortality with colonoscopy: what we know and what we don't know[END_REF] Prospective study 49 PPV for hyperplastic polyps, LGIEN, HGIEN and invasive cancer was 100%, 93.1%, 90.1% and 100% EC, endocytoscopy; PPV, positive predictive value; NPV, negative predictive value, LGIEN, low-grade intraepithelial neoplasia; HGIEN, high-grade intraepithelial neoplasia.


Tariq M Aslam

FRCSEd (Ophth), DM (OXON Waheeda Rahman

Peng David Henson

T Khaw

Frcophth David Henson

PhD, FBCO Peng T Khaw

Mr Tariq

M Aslam

A novel paediatric game-based visual fields assessor

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Measurement of visual fields can be prohibitively difficult in children, particularly below the age of 10-11 years. [START_REF] Wabbels | Feasibility and outcome of automated static perimetry in children using continuous light increment perimetry (CLIP) and fast threshold strategy[END_REF][START_REF] Marraffa | HPR perimetry and Humphrey perimetry in glaucomatous children[END_REF] Problems include lack of central fixation, [START_REF] Tschopp | Automated visual field examination in children aged 5-8 years. Part I: Experimental validation of a testing procedure[END_REF] short attention span [START_REF] Tschopp | Automated visual field examination in children aged 5-8 years. Part I: Experimental validation of a testing procedure[END_REF][START_REF] Blumenthal | The reliability of frequencydoubling perimetry in young children[END_REF][START_REF] Morales | The feasibility of short automated static perimetry in children[END_REF] and intolerance. Recent systems have demonstrated user interaction [START_REF] Marraffa | HPR perimetry and Humphrey perimetry in glaucomatous children[END_REF][START_REF] Martin | Rarebit and frequency-doubling technology perimetry in children and young adults[END_REF] may improve tolerance and reliability in children and have demonstrated the potential for personal computer testing. [START_REF] Damato | Multifixation campimetry on line: a perimeter for the detection of visual field loss using the internet[END_REF] Other recent innovations include eye tracking technology, proposed in 1989 [START_REF] Trope | Eye movement perimetry in glaucoma[END_REF] and developed at the University of Tokyo. [START_REF] Nunokawa | The influence of fixation on new visual field measurement using saccadic eye movement[END_REF] However, this requires expensive apparatus and has not yet been validated. [START_REF] Murray | Feasibility of saccadic vector optokinetic perimetry: a method of automated static perimetry for children using eye tracking[END_REF] Our aim was to determine whether a computer game system could be devised to measure visual fields in children whilst they play and enjoy an entertaining game.

Methods

The study had full ethics Committee approval and informed consent was obtained from all participants and parents. The development of the game-based visual fields assessor relied on multi-disciplinary input and combined international perimetric society standards [START_REF]Perimetry.Society. IPS Standards and guidelines[END_REF] with computer game science. The final system uses a supra-threshold examination strategy with fixed intensity. Background luminance and other psychophysical constants were set for this feasibility study based on literature review and estimations from adult trials of the test.

The software was developed using BBC basic /windows v5.80a and was conceived and written by the first author. Game design acknowledged Bateman and Boon's [START_REF] Bateman Cab | 21st Century Game Design[END_REF] game play types and Salen and Zimmerman [START_REF] Salen | Rules of Play: Game Design Fundamentals[END_REF] and Mulligan & Patrovsky 14 theories on player psychologies . We adopted the iterative game design process advocated by Salen and Zimmerman [START_REF] Salen | Rules of Play: Game Design Fundamentals[END_REF] and Fullerton et al., [START_REF] Fullerton | Games Design Workshop: Designing , Prototypingand Playtesting games[END_REF] which relies on gaining feedback from players early on and repeated game redesign -our game resulted from 17 cycles of appraisal and reprogramming. Development included designing instructions for explaining how to play the 'Crazy Castle' game and a cohesive storyline that ties in all that is required of the player.

The complete game structure consists of a large wooden castle (1m 3 ) with a set of occludable spectacle frames attached to a large central window on an exterior castle wall (fig. 1). A closed wooden drawbridge barrier obstructs view through the window into the castle. When a child's forehead is correctly aligned in the glasses (fig. 2,3) a switch activates opening of the drawbridge which then reveals a computer game on a screen (fig. 3) inside the castle, at a set distance from the spectacle frames. If the correct alignment is lost, the drawbridge instantly closes, hiding the game screen from view

The child uses an attached keypad to play the game, which is separated into four stages, allowing for the child to learn to play whilst maintaining position and for the software to set individualised variables. The game premise involves a small central character of a wizard with a plunger. When a tomato hovers under this plunger, the child presses the appropriately textured console button to squash the tomato (central fixation game). This central game is moved to different corners of the screen and peripheral field targets appear intermittently as ghosts in remaining corners. The child must continue the central fixation game but is encouraged to press a separate button when he sees these peripheral ghosts (peripheral target game). These ghosts (fig 4) appear at 10, 20 and 30 degrees from the central fixation game at each of 4 axes (see fig. 4 and5). They are presented for 200 milliseconds and increase in size if no response is provoked. Each tested peripheral target is graded 0 (not seen), 1 (5mm size seen ), 2 (4mm ), 3 ( 3mm ) or 4 ( 2mm). [START_REF] Henson | Visual Fields[END_REF] Missed stimuli are responded to with repeat testing. In accordance with perimetric standards and computer game theory, there are multiple programming measures, animations, sound effects and graphics incorporated to optimise central fixation importance, reduce false negatives and false positives and optimise utility of the game. The examiner is able to monitor progress on a separate laptop adjacent to the castle. Once tested, the children were invited to respond to a questionnaire about their experience with the visual field exam.

Results

19 children were recruited from an outpatient department at the Richard Desmond Children's Eye Centre at Moorfields Eye Hospital. Tests were carried out in the Fight for Sight NIHR Children's Research Facility. 2 children failed to complete the exam due to hardware and software failures that were subsequently corrected. 23 fields were completed, in 23 eyes of 17 children (6 bilateral). All completed fields had physiological blind spots found. Normal fields were defined through earlier pilot studies on normal adults. Median time for completion of the tests including training stages was 4.5 minutes (range 3.11 -8.0 minutes). There were 8 girls and 9 boys, aged 4 -14yrs (5 children aged 4-6, 8 aged 7-10 and 4 aged 10-14). Two four year olds with expected normal fields showed general delay in response which, according to our preset definitions, was classed as abnormal. The problems for these two children included, firstly, requiring a greater time to learn the game than was currently programmed into the system. Furthermore the requirement of two separate buttons to respond in the course of the game was perhaps too complex and a single button option might be more apt in this age group. However, it was very promising that they both still fully completed the tests, and we feel that reducing game difficulty for this age would counter the problems that arose. For all 15 other eyes with clinically expected normal fields (all normal eyes of children aged over 4), the children were, at their first attempt, able to complete the assessment giving normal visual fields. This included 3 children aged 6 and under. For the 5 eyes with expected glaucomatous loss, all 5 visual fields measured showed abnormal fields. Discrimination potential and acceptability of test are demonstrated in Fig. 4 and Fig. 5. In questionnaires, all children enjoyed playing the game and gave positive feedback.

Discussion

The gaming system has shown great promise, in this feasibility study, to provide clinically useful information in a way that is accessible and enjoyable for the child with no discomfort or potential for harm. Developments need to be made in terms of shortfalls in hardware and software as well as in optimising the psychophysical consistency and validity of peripheral targets. Ultimately we anticipate this system being particularly useful for perimetry in targeted groups of children in terms of age, aptitude, personality and physical ability. Formal validation and further reliability studies will be required before the system can be presented as a fully validated and reliable clinical tool. The final technology would potentially have a greater diagnostic precision and sensitivity to identify visual fields loss than existing tests. The test would be quicker and more reliable and therefore be a lesser drain on resources. It would be more affordable than alternative proposals in development and more enjoyable to experience for the child than any current system. It has potential for worldwide adoption due to its basis upon widely available and affordable technology.In modified form, the game-based system could also be utilised for other visual function measurements such as acuity and contrast sensitivity. Fields measured of 14 yr old boy with no ocular pathology in RE, but with glaucoma and suspected field defects found in his LE that corresponded with those found on Humphrey testing. Note in RE almost all targets were seen normally (score 3 or 4). In LE there are several areas of lost field (7 out of the 24 tested areas score less than 3). LE field was done after the RE Fig. 5 Acceptability of test. This 11 yr old child had documented poor compliance with standard fields tests and none were found acceptable. Goldman fields were not successful and Humphreys fields showed multiple areas of field loss with poor fixation. On his first attempt with our game-based fields analyser, however, he returned good peripheral fields whilst demonstrating good central target fixation, in accordance with his clinically expected visual function.

Game screen -visible inside castle

Fig. 1

 1 Fig. 1 Paediatric game-based vision assessor Fig. 2 Child waiting to play game, holding control pad then correctly positioned to see and play game Fig. 3 Examples of images seen in game; a) Tomato correctly positioned to mash, b) Tomato being mashed, c) peripheral ghost being lasered Fig. 4 Discrimination potential.Fields measured of 14 yr old boy with no ocular pathology in RE, but with glaucoma and suspected field defects found in his LE that corresponded with those found on Humphrey testing. Note in RE almost all targets were seen normally (score 3 or 4). In LE there are several areas of lost field (7 out of the 24 tested areas score less than 3). LE field was done after the RE

  fig 3(a)

Acknowledgements

This paper was funded by a research grant from the NIHR Biomedical Research Centre Moorfields Eye Hospital and UCL Institute of Ophthalmology and Moorfields Trustees. Support of the NIHR Manchester BRC is acknowledged. We would like to acknowledge input and comments from professionals including optometrists, ophthalmologists, vision scientists and paediatric nurses over the course of this study. Young members of the public also gave key input during the development phase of the game, with particularly useful and lengthy input from Humzah Jessa and Zoha Jessa.

Financial support: This paper was funded by a research grant from the NIHR Biomedical Research Centre Moorfields Eye Hospital and UCL Institute of Ophthalmology and Moorfields Trustees. The funding organisation had no role in the design or conduct of this research. No conflicting relationship exists for any author.


K Xiao

J Sliwka
email: jan.sliwka@ensta-bretagne.fr

L Jaulin

A wind-independent control strategy for autonomous sailboats based on Voronoi diagram

Keywords: robotics, autonomous sailboat, control, wind-independent, Voronoi diagram

This paper proposes a new control strategy based on Voronoi diagram for autonomous sailboats. This strategy applies the method of Voronoi diagram to decompose the space and uses a lookup table to make the boat sail as planned. The contribution of this paper is that for the first time it proposes an approach to control autonomous sailboats without the data from wind sensors (with data from GPS and compass only). Since the sensor for detecting wind direction can break down at any time, this new strategy, independent of wind direction, is much more reliable. A simulation is proposed to illustrate the principle and the effectiveness of the approach. Two real experiments on buggy and sailboat are also performed for validation.

Introduction

This paper puts up a new control strategy that is independent of wind sensor data for autonomous sailboats. It is motivated mainly by the microtransat challenge, which focuses on the development of small autonomous sailboat robots that could go cross the Atlantic ocean. The challenge is quite meaningful since the unmanned sailboats powered by wind have a great potential application in future oceanographic observations and long term offshore operations (see, [START_REF] Sliwka | Autonomous robotic boat of ensieta[END_REF] and 3 ).

Due to the harsh environment of the ocean, all components of such robots should be robust with respect to all situations (heavy weather, waves, salt water, low level of energy, long trip, . . . ). Sensors on autonomous sailboats could be divided into two types: One type is reliable sensors, which could survive to all situations. Such sensors include the GPS (providing the position and speed of the boat with a good accuracy), the compass (measuring the north direction with a rather good accuracy in ocean), the gyrometers (giving the rotational speed of the boat) and accelerometers (measuring the roll and pitch of the robot). All these sensors are low energy consumers, can be enclosed inside a waterproof tank and can survive for years. The other type is unreliable sensors, which have a high probability to break down in case of heavy weather. Anemometer (device that is used for measuring wind speed), weather vane (which returns the direction of the wind) and dynamometer (which measures the forces on the sail or the rudder) are considered as unreliable. They are directly in contact with aggressive natural elements (wind, waves, salt) and can fail at any time. For simplicity, anemometers and weather vane are called wind sensors in the following passage.

Traditionally, to control the boat, it is necessary to get data from unreliable sensors in order to know where the wind comes from and how strong it is (see e.g., [START_REF] Sauze | An autonomous sailing robot for ocean observation[END_REF] , 56 ). However, as said above, sensors for measuring the speed and direction of the wind are vulnerable and could not sustain for long periods ( 7). Therefore, we intend to take a new control strategy that could operate without the knowledge of wind direction and speed.

In this paper, we come up with a new and simple control strategy that is wind-independent based on the Voronoi diagram. We have noticed that for every sailboat there exists a certain no-go-angle θ no on the polar speed diagram ( 8 ). It means that no matter where the wind comes from, if we provide the sailboat with m (m ≥ 2) possible directions whose angular separations are greater than the no-go-angle, the sailboat could definitely follow one of them. Our new strategy is based on a lookup table which defines m (m ≥ 2) possible directions for each zone of the sea. We have introduced the method of Voronoi diagram to implement the decomposition of the sea. As a result, we don't need any data about the wind direction and speed to control our sailboat.

Section 2 provides the basics of polar speed diagram, Voronoi diagram and presents the sailboat to be considered. Section 3 explains how our control strategy works. Section 4 provides a simulation of the strategy. Two real tests on buggy and sailboat are presented on Section 5 and Section 6

Preliminaries

Polar Speed Diagram

As we know, if the boat is pointed too close to the wind (unless they are backed), the sails will be luffing ("flapping") in the breeze and cannot generate any effective power, only making noise like a flag. Different boats, of course, have different performance characteristics. These characteristics depend on such variables as hull design, keel design, rigging, sails, etc.

The polar diagram of a sailboat is the set S of all pairs (θ,v) that can be reached by the boat, in a cruising mode. The area of direction that cannot be reached is called a no-go-zone. The size of the no-go zone (no-goangle) will differ based on the performance characteristics of the particular sailboat. For example, racing sailboats can usually sail much closer to the wind (i.e., fewer degrees off the wind direction) than cruising yachts. This is known as "pointing higher."

However, for a given sailboat, the no-go-angle is certain and could be retrieved on its polar diagram. It means that the boat could definitely follow one of m (m ≥ 2) predefined directions that have an included angle greater than its no-go-angle, whatever the wind direction is.

Voronoi Diagram

A Voronoi diagram is a special kind of decomposition of a metric space determined by distances to a specified discrete set of objects in the space, e.g., by a discrete set of points. In the simplest case, we are given a set of points S in the plane, each point corresponding to one Voronoi site. Each point p has a Voronoi cell, V (p) consisting of all points closer to p than to any other point. The segments of the Voronoi diagram are all the points in the plane that are equidistant to the two nearest points.

In general, the set of all points closer to a point p of S than to any other point of S is the interior of a (in some cases unbounded) convex polytope, called the Dirichlet domain or Voronoi cell for p. The set of such polytopes tessellates the whole space, and is the Voronoi tessellation corresponding to the set S. If the dimension of the space is only 2, then it is easy to draw pictures of Voronoi tessellations, and in that case they are sometimes called Voronoi diagrams.

Given a set of S {p i ∈ R n }, the Voronoi cell associated with point p i is the set

V (p i ) {x | d(x, p i ) d(x, p j ), ∀i = j}
and the Voronoi diagram of the set S is given by the union of all of the Voronoi cells.

Another way to define V (p i ) is in terms of the intersection of halfplanes. Given two sites p i and p j , the set of points that are strictly closer to p i than to p j is just the open halfplane whose bounding line is the perpendicular bisector between p i and p j . Denote this halfplane h(p i , p j ). It is easy to see that a point q lies in V (p i ) if and only if q lies within the intersection of h(p i , p j ) for all j = i. In other words,

V (p i ) ∩h(p i , p j ), (∀j = i).
Since the intersection of halfplanes is a (possibly unbounded) convex polygon, it is easy to see that V (p i ) is a (possibly unbounded) convex polygon.

There are a number of algorithms for computing Voronoi diagrams. Of course, there is a simple O(n 2 log n) time algorithm, which operates by computing V (p i ) by intersecting all the bisector halfplanes h(p i , p j ),(∀j = i). However, there are much more efficient ways like the sweepline algorithm developed by Steven Fortune 9 , which has O(n log n) worst-case running time.

Voronoi diagram has already been applied on Robotics. For example, it was employed on a path planning strategy for mobile robots on a two-dimensional map, especially for avoidance of obstacles en route (see e.g. 10 ).

Sailboat

In order to simulate our control strategy, we need a sailboat dynamics model to implement it. We have introduced here the following state equations based on those in. [START_REF] Jaulin | Modlisation et commande d'un bateau 'a voile[END_REF] According to our measurement and test, they are a good approximation of the dynamics of the sailboat represented on Figure

2                                    ẋ = v cos θ + p 1 a cos ψ ẏ = v sin θ + p 1 a sin ψ θ = ω v =
fs sin δs-fr sin u1-p2v p9 ω = fs(p6-p7 cos δs)-p8fr cos u1-p3ω+p11 rand(t)

p10 f s = p 4 a sin (θ -ψ + δ s ) f r = p 5 v sin u 1 γ = cos (θ -ψ) + cos (u 2 ) δ s = π -θ + ψ if γ ≤ 0 sign (sin (θ -ψ)) .u 2 otherwise.
The state vector x = (x, y, θ, v, ω) T is composed with • the thrust force f s of wind on the sail • the force f r of water on the rudder • the maximum possible angle of the sail δ s at the current length of mainsheet (γ is to judge whether the sail is tight or not)

The parameters p i , a, ψ are assumed to be known exactly. They are

• the drift coefficient p 1
• the tangential friction coefficient p 2 between the boat and water • the angular friction coefficient p 3 between the boat and water • the sail lift coefficient p 4 and the rudder lift coefficient p 5 • p 6 , p 7 , p 8 could easily be seen from Figure 2 • the mass of the boat p 9 and the angular inertia of the boat p 10 The sailboat has two inputs: u 1 = δ r is the angle between the rudder and the sailboat and u 2 is the current length of the mainsheet which limits δ s . This model is similar to that described in, 11 except that here, (i) the control is not any more the sail angle, but the length of the mainsheet, which is more realistic, (ii) we added a random input on ω that represents the random wave effect on the direction of the boat.

It should be noted that these state equations are introduced only for simulation and presentation purpose. They are not assumed as excellent models, just serving as an example. Any other dynamic models for sailboat could also be applied. Moreover, we simulate the behavior of the boat using Euler approximation.

Control Strategy

Algorithm

Our control algorithm is divided into two parts, one offline and the other online. The offline part is performed by the PC and needs human intervention while the online part is done by the microcontroller embedded in the sailboat.

The offline part

The offline part is mainly a planning step and needs human help. It takes time to draw the Voronoi diagram with our current technique and thus it is not suitable to be performed on the microcontroller. The PC could do the job and gives out the computed Voronoi diagram to the embedded microcontroller. The offline part is performed at the beginning of one certain voyage.

step1: According to start and end points and obstacles en route, decompose the space by giving reference points that define the Voronoi diagram.

step2: Compute the Voronoi diagram. step3: Assign each Voronoi site with m (m ≥ 2) directions. step4: Generate the C code for the microcontroller.

Remark 3.1. The angular separations between the m directions in one Voronoi site must be greater than the no-go-angle of the boat in order that the boat could follow at least one of them.

Remark 3.2. The effectiveness and efficiency of the strategy depends largely on the setting of reference points and directions. It needs to be done by an experienced sailor.

Remark 3.3. All the directions should ultimately lead to the target in some way.

Remark 3.4. To avoid obstacles some reference points should appear before them and directions associated with these sites should point outward from the obstacles.

The online part

The online part of the algorithm is the action step and performed by the embedded microcontroller. It takes the Voronoi diagram given by the PC and controls the action of the sailboat in real-time. The steps are as follows:

step1: Check which Voronoi site the sailboat is in. step2: Choose the first direction assigned to the current site. step3: Tune the sail so that the boat gets maximum speed on the target direction and control the rudder so that the boat goes on the target direction.

step4: If the boat fails to follow the current given direction, choose the next one of the current site.

step5: Go to step3. step6: If it fails again, go to step2.

Remark 3.5. To determine whether the sailboat follows the desired direction, there could be many criteria using the sensor data about speed, direction or position of the boat. After comparison with other means, we chose the most well-proven way combining the data from GPS sensor and compass which are quite accurate in our scale of problem. The criteria is that if the actual direction of the boat during our observation time (the current position with respect to the last one observed) is within a certain zone around the target direction, then we assume the boat is successful in following it (shown on Figure 3).

Tuning the Sail

Because the wind direction is totally unknown, we cannot rely on any existing knowledge to control the angle of sail. As a result, we need to tune the sail to find the best sail angle so that the boat could fully utilize the power of the wind and follow the given direction. In our current strategy, we have two tuning functions. The first is to find the global optimal angle through a traversal technique, namely it tries every possible angle of sail and finds the best one. This function will be called when the given direction is changed, which means a totally different wind direction with respect to the boat will appear and therefore the best sail angle would be quite different from the last one. The second is to change the sail angle a little once at a time to try to find the local optimal angle. This function helps maintain the stability of the boat while searching for the best sail angle and avoids the high cost of time the first one needs.

Controlling the Boat Direction

In our control strategy, it is quite important for the boat to follow the given direction. For that purpose, a simple but efficient method for controlling the rudder should be applied, since the sailboat's direction is controlled mainly by the rudder. For most of the sailboats, the rudder is efficient enough to make the boat follow the target direction as long as it doesn't belong to the no-go-zone of polar diagram. In our current strategy, the controller for the rudder is one kind of proportional (proportional to the difference between desired direction and current boat direction). Figure 4 shows how the controller works with different target directions.

Simulation

Applying the control strategy and the sailboat model on SCILAB, we conducted a simulation of the new control strategy. We chose the Brest Gulf as our simulation environment because we are familiar with its conditions. Again, it only serves to present our strategy and any other environment could be chosen for the test. We imported the boundary of Brest Gulf into the simulation program (the black lines represent the shoreline). According to our destination and desired path, we decomposed the map into 6 Voronoi sites (the red lines represent the boundary of Voronoi zones) and set the corresponding reference points (the red circles in the figure) along the desired path. To simplify the simulation without losing generality, we assigned each site with only two given directions (the two arrows associated with each reference point, with the darker one representing the first direction and the lighter one representing the second direction) that point to the destination in some way and make the boat avoid obstacles. Since the no-go-angle for most sailboats (including ours [START_REF] Sliwka | Autonomous robotic boat of ensieta[END_REF] ) is less than 60 degrees, the included angle between the two directions of each Voronoi site should be greater than 60 degrees. The boat is represented by the black polygon.

For simplicity, we used the straightforward algorithm previously described in Section 2.2: T .

V (p i ) ∩h(p i , p j ), (∀j = i)
The result is satisfactory: the boat goes from the starting point to the target without hitting obstacles in any given wind direction. One trajectory of the boat is shown on Figure 5.

The SCILAB code of the simulation as well as movies illustrating our simulation can be downloaded at http://www.ensieta.fr/jaulin/kai.html 5. Testcase

With Buggy

In order to easily implement and see the result of our new control strategy, we tested first on a general ground robotic platform called buggy. There are some small differences between controlling the buggy and the sailboat since the buggy could follow any direction without difficulty. So we ignore the step3 and step5 of the online part that tune the sail. Instead, we just give the buggy a fixed speed. The steering servo which controls the direction of the buggy could be controlled as the rudder of the sailboat. And if we need to see what would happen when it fails to follow one direction, we Fig. 6. buggy could just move it in the wrong direction by hand. We chose a football field for test so that the GPS data would be quite accurate. At first, we intended the buggy to go around the field following some kind of spiral trajectory that directs the buggy to the center of the field. So we chose 4 reference points and decomposed the field into 4 sites. The sites and directions belonging to each site could be seen on the left half of Figure 7. The actual trajectory of the buggy is shown on the right half of the figure. The result was quite satisfactory, with the buggy following given direction quite well and it followed a spiral trajectory on the field as intended.

With Sailboat

After satisfactory results on buggy, we did a real test with our sailboat on Lake Tycolo near Brest, France. Further information about our sailboat could be seen in. [START_REF] Sliwka | Autonomous robotic boat of ensieta[END_REF] The only difference is that we unmounted the wind sensor (see Figure 8). Lake Tycolo is a small lake with good eyesight so that the behavior of the sailboat could be better watched during the test. For our test we intended the boat to travel from the bank of lake to the middle of it without hitting any boundary of the lake. According to our strategy, we decomposed the lake into 4 Voronoi sites. Again for simplicity, we assigned each site with only two directions whose angular seperation is greater than no-go-angle (60 degrees for our testcase). Then, we implemented the online part of the control strategy described above on a PIC microcontroller embedded in our sailboat.

The result of the test is quite satisfactory. The boat could tune its sail so that it could find the best sail angle for the moment, although it does not have any information about the wind. The angle the boat finds by itself • The strategy's biggest advantage is that it is wind-independent, thus much more reliable. As shown above, since the boat could definitely follow one of the two given directions that ultimately point to the target, the boat will undoubtedly get to the target no matter where the wind comes from. Thus, the strategy would be much more reliable than most of others that depend highly on the data from wind sensors which are not so reliable especially in harsh environment. And due to the simplicity of the strategy, the new strategy would have little chance to fail. • The strategy is simple and easy to implement. Due to the Voronoi diagram and the look-up table method, the strategy is quite simple and can be applied easily without putting a high demand on the hardware. Moreover, thanks to the simplicity of the strategy, the program costs few time to execute. In our real test for the sailboat, the computation time for one loop is at the millisecond level. With some advanced table look-up method and algorithms for generating Voronoi diagram, it could be even faster.

Although the strategy has been illustrated only on sailboats, it could have many other areas of application where some unreliable data are nec-essary for a reliable control. Some potential application areas include space exploration robots, autonomous submarines, etc. Some future work could help improve the strategy:

• Although the strategy works in our test, its effectiveness and efficiency depend largely on the quality of Voronoi diagram and the two given directions assigned to each Voronoi site. As a result, we currently need an experienced sailor to construct the Voronoi diagram and assign the two given directions. This would greatly limit its application. Future work could focus on finding some techniques to generate the Voronoi diagrams automatically. Many methods such as fuzzy logic, expert system and evolutionary algorithms could be turned for and there are a lot of interesting topics to discover. • Another important improvement for our strategy is on the computation of Voronoi diagram. To compute the Voronoi diagram takes time. Our current solution is to compute it once and for all in the initialization stage of the program and store it in memory. But it has brought about another problem: in this way the Voronoi diagram could not be updated in real-time. Although the problem does not matter in most cases, it would limit the application of the strategy in some areas. The problem could be largely mitigated by applying some advanced algorithms for computing Voronoi diagram. Now we used a simple, reliable but time-consuming algorithm. There exist a great number of more efficient algorithms for computing Voronoi diagram and Delaunay triangulation. Applying them in our strategy could largely reduce the initialization time for the whole system and could even incorporate the computation in every loop so that the Voronoi diagram could be updated in real time.

Fig. 1 .

 1 Fig. 1. sailboat with wind sensor

•

  the coordinates x, y of the inertial center G of the boat • the orientation θ of the boat • the tangential speed v of G • the angular velocity ω of the boat around G The internal variables are

Fig. 2 .

 2 Fig. 2. Sailboat to be observed

Fig. 3 .

 3 Fig. 3. a) the boat doesn't follow desired direction b) the boat follows target direction

Fig. 4 .

 4 Fig. 4. rudder control strategy

Fig. 5 .

 5 Fig. 5. simulation result

Fig. 7 .

 7 Fig. 7. test with the buggy

Fig. 9 .

 9 Fig. 9. test with the sailboat robot