The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Melissa C Skala - One of the best experts on this subject based on the ideXlab platform.
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Autofluorescence Imaging of 3d tumor macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism
Cancer Research, 2020Co-Authors: Tiffany M Heaster, Mouhita Humayun, David J Beebe, Melissa C SkalaAbstract:Macrophages within the tumor microenvironment (TME) exhibit a spectrum of protumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate two-photon Autofluorescence Imaging of NAD(P)H and FAD to nondestructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours poststimulation for mouse macrophages (RAW264.7) stimulated with IFNγ or IL4 plus IL13 in 2D culture, confirming that Autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse polyoma-middle T virus or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor cocultures exhibited significantly different FAD mean lifetimes and greater migration than monocultures at 24, 48, and 72 hours postseeding. In cocultures with primary human cancer cells, actively migrating monocyte-derived macrophages had greater redox ratios [NAD(P)H/FAD intensity] compared with passively migrating monocytes at 24 and 48 hours postseeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free Autofluorescence Imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and Imaging system provides unique insights into spatiotemporal tumor-immune cross-talk within the 3D TME. SIGNIFICANCE: Label-free metabolic Imaging and microscale culture technologies enable monitoring of single-cell macrophage metabolism, migration, and function in the 3D tumor microenvironment.
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Autofluorescence Imaging of 3d tumor macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism
bioRxiv, 2020Co-Authors: Tiffany M Heaster, Mouhita Humayun, David J Beebe, Melissa C SkalaAbstract:Macrophages within the tumor microenvironment (TME) exhibit a spectrum of pro-tumor and anti-tumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate two-photon Autofluorescence Imaging of NAD(P)H and FAD to non-destructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours post-stimulation for mouse macrophages (RAW 264.7) stimulated with IFN-{gamma} or IL-4 plus IL-13 in 2D culture, validating that Autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse PyVMT or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor co-cultures exhibited significantly different FAD mean lifetimes and greater migration than mono-cultures at 24, 48, and 72 hours post-seeding. In co-cultures with primary human cancer cells, actively-migrating monocyte-derived macrophages had greater redox ratios (NAD(P)H/FAD intensity) compared to passively-migrating monocytes at 24 and 48 hours post-seeding, reflecting metabolic heterogeneity in this sub-population of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free Autofluorescence Imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and Imaging system provides unique insights into spatiotemporal tumor-immune crosstalk within the 3D TME.
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Autofluorescence Imaging identifies tumor cell cycle status on a single cell level
Journal of Biophotonics, 2018Co-Authors: Tiffany M Heaster, Melissa C Skala, Alex J Walsh, Yue Zhao, Scott W HiebertAbstract:The goal of this study is to validate fluorescence intensity and lifetime Imaging of metabolic co-enzymes NAD(P)H and FAD (optical metabolic Imaging, or OMI) as a method to quantify cell-cycle status of tumor cells. Heterogeneity in tumor cell-cycle status (e. g. proliferation, quiescence, apoptosis) increases drug resistance and tumor recurrence. Cell-cycle status is closely linked to cellular metabolism. Thus, this study applies cell-level metabolic Imaging to distinguish proliferating, quiescent, and apoptotic populations. Two-photon microscopy and time-correlated single photon counting are used to measure optical redox ratio (NAD(P)H fluorescence intensity divided by FAD intensity), NAD(P)H and FAD fluorescence lifetime parameters. Redox ratio, NAD(P)H and FAD lifetime parameters alone exhibit significant differences (p<0.05) between population means. To improve separation between populations, linear combination models derived from partial least squares - discriminant analysis (PLS-DA) are used to exploit all measurements together. Leave-one-out cross validation of the model yielded high classification accuracies (92.4 and 90.1 % for two and three populations, respectively). OMI and PLS-DA also identifies each sub-population within heterogeneous samples. These results establish single-cell analysis with OMI and PLS-DA as a label-free method to distinguish cell-cycle status within intact samples. This approach could be used to incorporate cell-level tumor heterogeneity in cancer drug development.
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in vivo Autofluorescence Imaging of tumor heterogeneity in response to treatment
Neoplasia, 2015Co-Authors: Amy T Shah, Kirsten E Diggins, Alex J Walsh, Jonathan M Irish, Melissa C SkalaAbstract:Subpopulations of cells that escape anti-cancer treatment can cause relapse in cancer patients. Therefore, measurements of cellular-level tumor heterogeneity could enable improved anti-cancer treatment regimens. Cancer exhibits altered cellular metabolism, which affects the Autofluorescence of metabolic cofactors NAD(P)H and FAD. The optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD) reflects global cellular metabolism. The fluorescence lifetime (amount of time a fluorophore is in the excited state) is sensitive to microenvironment, particularly protein-binding. High-resolution Imaging of the optical redox ratio and fluorescence lifetimes of NAD(P)H and FAD (optical metabolic Imaging) enables single-cell analyses. In this study, mice with FaDu tumors were treated with the antibody therapy cetuximab or the chemotherapy cisplatin and imaged in vivo two days after treatment. Results indicate that fluorescence lifetimes of NAD(P)H and FAD are sensitive to early response (two days post-treatment, P<.05), compared with decreases in tumor size (nine days post-treatment, P<.05). Frequency histogram analysis of individual optical metabolic Imaging parameters identifies subpopulations of cells, and a new heterogeneity index enables quantitative comparisons of cellular heterogeneity across treatment groups for individual variables. Additionally, a dimensionality reduction technique (viSNE) enables holistic visualization of multivariate optical measures of cellular heterogeneity. These analyses indicate increased heterogeneity in the cetuximab and cisplatin treatment groups compared with the control group. Overall, the combination of optical metabolic Imaging and cellular-level analyses provide novel, quantitative insights into tumor heterogeneity.
K Singh - One of the best experts on this subject based on the ideXlab platform.
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smartphone based multimodal tethered capsule endoscopic platform for white light narrow band and fluorescence Autofluorescence Imaging
Journal of Biophotonics, 2021Co-Authors: Gargi Sharma, Oanamaria Thoma, Katharina Blessing, Robert Gal, Maximilian J Waldner, K SinghAbstract:Multimodal low-cost endoscopy is highly desirable in poor resource settings such as in developing nations. In this work, we developed a smartphone-based low-cost, reusable tethered capsule endoscopic platform that allows white-light, narrowband, and fluorescence/Autofluorescence Imaging of the esophagus. The ex-vivo studies of swine esophagus were performed and compared with a commercial endoscope to test the white-light Imaging capabilities of the endoscope. The efficacy of the capsule for narrow-band Imaging was tested by Imaging the vascularization of the tongue. To determine the Autofluorescence/fluorescence capability of the endoscope, fluorescein dye with different concentrations was imaged. Furthermore, swine esophagus injected with fluorescein dye was imaged using the fluorescence/Autofluorescence and the white-light Imaging modules, ex-vivo. The overall cost of the capsules is approximately 12 €, 15 €, and 42 € for the white light Imaging, the narrow-band Imaging, and the fluorescence/Autofluorescence Imaging respectively. In addition, the cost of the laser source module required for the narrow-band Imaging and the fluorescence/Autofluorescence Imaging is approximately 218 €. This device will open the possibility of Imaging the esophagus in underprivileged areas.
Tiffany M Heaster - One of the best experts on this subject based on the ideXlab platform.
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Autofluorescence Imaging of 3d tumor macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism
Cancer Research, 2020Co-Authors: Tiffany M Heaster, Mouhita Humayun, David J Beebe, Melissa C SkalaAbstract:Macrophages within the tumor microenvironment (TME) exhibit a spectrum of protumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate two-photon Autofluorescence Imaging of NAD(P)H and FAD to nondestructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours poststimulation for mouse macrophages (RAW264.7) stimulated with IFNγ or IL4 plus IL13 in 2D culture, confirming that Autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse polyoma-middle T virus or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor cocultures exhibited significantly different FAD mean lifetimes and greater migration than monocultures at 24, 48, and 72 hours postseeding. In cocultures with primary human cancer cells, actively migrating monocyte-derived macrophages had greater redox ratios [NAD(P)H/FAD intensity] compared with passively migrating monocytes at 24 and 48 hours postseeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free Autofluorescence Imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and Imaging system provides unique insights into spatiotemporal tumor-immune cross-talk within the 3D TME. SIGNIFICANCE: Label-free metabolic Imaging and microscale culture technologies enable monitoring of single-cell macrophage metabolism, migration, and function in the 3D tumor microenvironment.
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Autofluorescence Imaging of 3d tumor macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism
bioRxiv, 2020Co-Authors: Tiffany M Heaster, Mouhita Humayun, David J Beebe, Melissa C SkalaAbstract:Macrophages within the tumor microenvironment (TME) exhibit a spectrum of pro-tumor and anti-tumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate two-photon Autofluorescence Imaging of NAD(P)H and FAD to non-destructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours post-stimulation for mouse macrophages (RAW 264.7) stimulated with IFN-{gamma} or IL-4 plus IL-13 in 2D culture, validating that Autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse PyVMT or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor co-cultures exhibited significantly different FAD mean lifetimes and greater migration than mono-cultures at 24, 48, and 72 hours post-seeding. In co-cultures with primary human cancer cells, actively-migrating monocyte-derived macrophages had greater redox ratios (NAD(P)H/FAD intensity) compared to passively-migrating monocytes at 24 and 48 hours post-seeding, reflecting metabolic heterogeneity in this sub-population of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free Autofluorescence Imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and Imaging system provides unique insights into spatiotemporal tumor-immune crosstalk within the 3D TME.
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Autofluorescence Imaging identifies tumor cell cycle status on a single cell level
Journal of Biophotonics, 2018Co-Authors: Tiffany M Heaster, Melissa C Skala, Alex J Walsh, Yue Zhao, Scott W HiebertAbstract:The goal of this study is to validate fluorescence intensity and lifetime Imaging of metabolic co-enzymes NAD(P)H and FAD (optical metabolic Imaging, or OMI) as a method to quantify cell-cycle status of tumor cells. Heterogeneity in tumor cell-cycle status (e. g. proliferation, quiescence, apoptosis) increases drug resistance and tumor recurrence. Cell-cycle status is closely linked to cellular metabolism. Thus, this study applies cell-level metabolic Imaging to distinguish proliferating, quiescent, and apoptotic populations. Two-photon microscopy and time-correlated single photon counting are used to measure optical redox ratio (NAD(P)H fluorescence intensity divided by FAD intensity), NAD(P)H and FAD fluorescence lifetime parameters. Redox ratio, NAD(P)H and FAD lifetime parameters alone exhibit significant differences (p<0.05) between population means. To improve separation between populations, linear combination models derived from partial least squares - discriminant analysis (PLS-DA) are used to exploit all measurements together. Leave-one-out cross validation of the model yielded high classification accuracies (92.4 and 90.1 % for two and three populations, respectively). OMI and PLS-DA also identifies each sub-population within heterogeneous samples. These results establish single-cell analysis with OMI and PLS-DA as a label-free method to distinguish cell-cycle status within intact samples. This approach could be used to incorporate cell-level tumor heterogeneity in cancer drug development.
Pierre Lane - One of the best experts on this subject based on the ideXlab platform.
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endoscopic high resolution Autofluorescence Imaging and oct of pulmonary vascular networks
Optics Letters, 2016Co-Authors: Hamid Pahlevaninezhad, Anthony M D Lee, Geoffrey Hohert, Tawimas Shaipanich, Stephen Lam, Calum Macaulay, Evelea Beaudoin, Caroline Boudoux, Pierre LaneAbstract:High-resolution Imaging from within airways may allow new methods for studying lung disease. In this work, we report an endoscopic Imaging system capable of high-resolution Autofluorescence Imaging (AFI) and optical coherence tomography (OCT) in peripheral airways using a 0.9 mm diameter double-clad fiber (DCF) catheter. In this system, AFI excitation light is coupled into the core of the DCF, enabling tightly focused excitation light while maintaining efficient collection of Autofluorescence emission through the large diameter inner cladding of the DCF. We demonstrate the ability of this Imaging system to visualize pulmonary vasculature as small as 12 μm in vivo.
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pulmonary nodule and vasculature detection in vivo using endoscopic doppler optical coherence tomography and Autofluorescence Imaging
Cancer, 2016Co-Authors: Hamid Pahlevaninezhad, Anthony M D Lee, Geoffrey Hohert, Tawimas Shaipanich, Alexander Ritchie, Wei Zhang, Stephen Lam, Calum Macaulay, Pierre LaneAbstract:This work presents endoscopic Doppler optical coherence tomography and Autofluorescence Imaging (DOCT-AFI) of peripheral pulmonary nodules and vascular networks in vivo using a small 0.9 mm diameter fiber catheter.
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endoscopic doppler optical coherence tomography and Autofluorescence Imaging of peripheral pulmonary nodules and vasculature
Biomedical Optics Express, 2015Co-Authors: Hamid Pahlevaninezhad, Anthony M D Lee, Geoffrey Hohert, Tawimas Shaipanich, Alexander Ritchie, Wei Zhang, Stephen Lam, Calum Macaulay, Diana N Ionescu, Pierre LaneAbstract:We present the first endoscopic Doppler optical coherence tomography and co-registered Autofluorescence Imaging (DOCT-AFI) of peripheral pulmonary nodules and vascular networks in vivo using a small 0.9 mm diameter catheter. Using exemplary images from volumetric data sets collected from 31 patients during flexible bronchoscopy, we demonstrate how DOCT and AFI offer complementary information that may increase the ability to locate and characterize pulmonary nodules. AFI offers a sensitive visual presentation for the rapid identification of suspicious airway sites, while co-registered OCT provides detailed structural information to assess the airway morphology. We demonstrate the ability of AFI to visualize vascular networks in vivo and validate this finding using Doppler and structural OCT. Given the advantages of higher resolution, smaller probe size, and ability to visualize vasculature, DOCT-AFI has the potential to increase diagnostic accuracy and minimize bleeding to guide biopsy of pulmonary nodules compared to radial endobronchial ultrasound, the current standard of care.
Evelien Dekker - One of the best experts on this subject based on the ideXlab platform.
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diagnostic performance of narrowed spectrum endoscopy Autofluorescence Imaging and confocal laser endomicroscopy for optical diagnosis of colonic polyps a meta analysis
Lancet Oncology, 2013Co-Authors: Linda K Wanders, S E Uitentuis, Mariska M G Leeflang, James E East, Evelien DekkerAbstract:Summary Background Novel endoscopic technologies could allow optical diagnosis and resection of colonic polyps without histopathological testing. Our aim was to establish the sensitivity, specificity, and real-time negative predictive value of three types of narrowed spectrum endoscopy (narrow-band Imaging [NBI], image-enhanced endoscopy [i-scan], and Fujinon intelligent chromoendoscopy [FICE]), confocal laser endomicroscopy (CLE), and Autofluorescence Imaging for differentiation between neoplastic and non-neoplastic colonic lesions. Methods We identified relevant studies through a search of Medline, Embase, PubMed, and the Cochrane Library. Clinical trials and observational studies were eligible for inclusion when the diagnostic performance of NBI, i-scan, FICE, Autofluorescence Imaging, or CLE had been assessed for differentiation, with histopathology as the reference standard, and for which a 2 × 2 contingency table of lesion diagnosis could be constructed. We did a random-effects bivariate meta-analysis using a non-linear mixed model approach to calculate summary estimates of sensitivity and specificity, and plotted estimates in a summary receiver-operating characteristic curve. Findings We included 91 studies in our analysis: 56 were of NBI, ten of i-scan, 14 of FICE, 11 of CLE, and 11 of Autofluorescence Imaging (more than one of the investigated modalities assessed in eight studies). For NBI, overall sensitivity was 91·0% (95% CI 88·6–93·0), specificity 85·6% (81·3–89·0), and real-time negative predictive value 82·5% (75·4–87·9). For i-scan, overall sensitivity was 89·3% (83·3–93·3), specificity 88·2% (80·3–93·2), and real-time negative predictive value 86·5% (78·0–92·1). For FICE, overall sensitivity was 91·8% (87·1–94·9), specificity 83·5% (77·2–88·3), and real-time negative predictive value 83·7% (77·5–88·4). For Autofluorescence Imaging, overall sensitivity was 86·7% (79·5–91·6), specificity 65·9% (50·9–78·2), and real-time negative predictive value 81·5% (54·0–94·3). For CLE, overall sensitivity was 93·3% (88·4–96·2), specificity 89·9% (81·8–94·6), and real-time negative predictive value 94·8% (86·6–98·1). Interpretation All endoscopic Imaging techniques other than Autofluorescence Imaging could be used by appropriately trained endoscopists to make a reliable optical diagnosis for colonic lesions in daily practice. Further research should be focused on whether training could help to improve negative predictive values. Funding None.
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Hyperplastic polyposis syndrome: a pilot study for the differentiation of polyps by using high-resolution endoscopy, Autofluorescence Imaging, and narrow-band Imaging
Gastrointestinal endoscopy, 2009Co-Authors: Karam S. Boparai, Paul Fockens, Frank J.c. Van Den Broek, Susanne Van Eeden, Evelien DekkerAbstract:Background Endoscopic differentiation and removal of potentially premalignant sessile serrated adenomas (SSAs) may be important steps in preventing the development of colorectal cancer in hyperplastic polyposis syndrome (HPS). Objective To assess the value of high-resolution endoscopy, Autofluorescence Imaging (AFI), and narrow-band Imaging (NBI) for differentiating polyps in HPS. Design A prospective polyp series. Setting Single tertiary referral center. Patients and Interventions Seven patients with HPS underwent colonoscopy with endoscopic trimodal Imaging, which incorporates high-resolution endoscopy, AFI, and NBI in 1 system. All detected polyps were analyzed with AFI for color and with NBI for Kudo pit pattern and vascular pattern intensity. Main Outcome Measurements The accuracy, sensitivity, and specificity of AFI and NBI in differentiating detected polyps were determined by using histology as the criterion standard. Results A total of 19 hyperplastic polyps (HPs), 32 SSAs, and 15 adenomas were detected. For differentiating SSAs from HPs, AFI color, Kudo pit pattern, and vascular pattern intensity resulted in a diagnostic accuracy of 55%, 55%, and 52%, respectively. For differentiating adenomas from HPs, the accuracy was 65%, 94%, and 90%, respectively. Macroscopically, the combination of a size of 3 mm or larger and a proximal location resulted in the highest accuracy (76%) for differentiating SSAs from HPs. Limitation Small sample size. Conclusion Endoscopic differentiation between HPs and SSAs by using endoscopic trimodal Imaging proved unsatisfactory. Differentiation of adenomas from HPs was possible with NBI but not with AFI.
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endoscopic tri modal Imaging for surveillance in ulcerative colitis randomised comparison of high resolution endoscopy and Autofluorescence Imaging for neoplasia detection and evaluation of narrow band Imaging for classification of lesions
Gut, 2008Co-Authors: F. J. C. Van Den Broek, Paul Fockens, S. Van Eeden, Johannes B Reitsma, James C H Hardwick, Pieter C F Stokkers, Evelien DekkerAbstract:Background: Endoscopic tri-modal Imaging (ETMI) incorporates white light endoscopy (WLE), Autofluorescence Imaging (AFI) and narrow-band Imaging (NBI). Aims: To assess the value of ETMI for the detection and classification of neoplasia in patients with longstanding ulcerative colitis. Design: Randomised comparative trial of tandem colonoscopies. Setting: Academic Medical Centre Amsterdam, Netherlands. Patients and methods: Fifty patients with ulcerative colitis underwent surveillance colonoscopy with ETMI. Each colonic segment was inspected twice, once with AFI and once with WLE, in random order. All detected lesions were inspected by NBI for Kudo pit pattern analysis and additional random biopsies were taken. Main outcome measures: Neoplasia miss-rates of AFI and WLE, and accuracy of the Kudo classification by NBI. Results: Among patients assigned to inspection with AFI first (n = 25), 10 neoplastic lesions were primarily detected. Subsequent WLE detected no additional neoplasia. Among patients examined with WLE first (n = 25), three neoplastic lesions were detected; subsequent inspection with AFI added three neoplastic lesions. Neoplasia miss-rates for AFI and WLE were 0% and 50% (p = 0.036). The Kudo classification by NBI had a sensitivity and specificity of 75% and 81%; however, all neoplasia was coloured purple on AFI (sensitivity 100%). No additional patients with neoplasia were detected by random biopsies. Conclusion: Autofluorescence Imaging improves the detection of neoplasia in patients with ulcerative colitis and decreases the yield of random biopsies. Pit pattern analysis by NBI has a moderate accuracy for the prediction of histology, whereas AFI colour appears valuable in excluding the presence of neoplasia. Trial registration number: ISRCTN05272746
