Multiview confocal super-resolution microscopy.

Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.

A reference library for assigning protein subcellular localizations by image-based machine learning.

Confocal micrographs of EGFP fusion proteins localized at key cell organelles in murine and human cells were acquired for use as subcellular localization landmarks. For each of the respective 789,011 and 523,319 optically validated cell images, morphology and statistical features were measured. Machine learning algorithms using these features permit automated assignment of the localization of other proteins and dyes in both cell types with very high accuracy. Automated assignment of subcellular localizations for model tail-anchored proteins with randomly mutated C-terminal targeting sequences allowed the discovery of motifs responsible for targeting to mitochondria, endoplasmic reticulum, and the late secretory pathway. Analysis of directed mutants enabled refinement of these motifs and characterization of protein distributions in within cellular subcompartments.

Advanced imaging in surveillance of Barrett's esophagus: Is the juice worth the squeeze?

Esophageal cancer is on the rise. The known precursor lesion is Barrett's esophagus (BE). Patients with dysplasia are at higher risk of developing esophageal cancer. Currently the gold standard for surveillance endoscopy involves taking targeted biopsies of abnormal areas as well as random biopsies every 1-2 cm of the length of the Barrett's. Unfortunately studies have shown that this surveillance can miss dysplasia and cancer. Advanced imaging technologies have been developed that may help detect dysplasia in BE. This opinion review discusses advanced imaging in BE surveillance endoscopy and its utility in clinical practice.

Development and Validation of Confocal Endomicroscopy Diagnostic Criteria for Low-Grade Dysplasia in Barrett's Esophagus.

OBJECTIVES: Low-grade dysplasia (LGD) in Barrett's esophagus (BE) is generally inconspicuous on conventional and magnified endoscopy. Probe-based confocal laser endomicroscopy (pCLE) provides insight into gastro-intestinal mucosa at cellular resolution. We aimed to identify endomicroscopic features and develop pCLE diagnostic criteria for BE-related LGD. METHODS: This was a retrospective study on pCLE videos generated in 2 prospective studies. In phase I, 2 investigators assessed 30 videos to identify LGD endomicroscopic features, which were then validated in an independent video set (n = 25). Criteria with average accuracy >80% and interobserver agreement κ > 0.4 were taken forward. In phase II, 6 endoscopists evaluated the criteria in an independent video set (n = 57). The area under receiver operating characteristic curve was constructed to find the best cutoff. Sensitivity, specificity, interobserver, and intraobserver agreements were calculated. RESULTS: In phase I, 6 out of 8 criteria achieved the agreement and accuracy thresholds (i) dark nonround glands, (ii) irregular gland shape, (iii) lack of goblet cells, (iv) sharp cutoff of darkness, (v) variable cell size, and (vi) cellular stratification. The best cutoff for LGD diagnosis was 3 out of 6 positive criteria. In phase II, the diagnostic criteria had a sensitivity and specificity for LGD of 81.9% and 74.6%, respectively, with an area under receiver operating characteristic of 0.888. The interobserver agreement was substantial (κ = 0.654), and the mean intraobserver agreement was moderate (κ = 0.590). CONCLUSIONS: We have generated and validated pCLE criteria for LGD in BE. Using these criteria, pCLE diagnosis of LGD is reproducible and has a substantial interobserver agreement.

Individual probe based confocal laser endomicroscopy criteria in the analysis of indeterminate biliary strictures.

OBJECTIVES: Evaluation of indeterminate biliary strictures remains challenging due to limited sensitivity of endoscopic tissue sampling. Biliary probe-based confocal laser endomicroscopy (pCLE) has shown promise to detect and exclude neoplasia. However, knowledge of whether individual inflammatory criteria are more prevalent in neoplasia compared to benign strictures is limited. The objective of this work is to improve diagnosis of neoplastic and inflammatory conditions using pCLE. MATERIALS AND METHODS: The charts of all patients who underwent pCLE at a single referral center between 2009 and 2015 were reviewed. ERCP reports were reviewed for eleven Miami and Paris criteria. Primary outcome was the identification of neoplasia by histopathology (defined as high-grade dysplasia and/or adenocarcinoma). To model predictors of neoplasia, we fit a binary regression model incorporating data from pCLE operating criteria, pCLE impression, and PSC status. RESULTS: 97 patients were identified. In the 27 patients with neoplasia, there was increasing number of Miami malignant criteria (Pearson r = 0.512, p < .001) while inflammatory criteria were less prevalent. 10% (5/51, p < .001) of patients with benign pCLE impression developed neoplasia, while 48% (22/46, p < .001) with suspicious pCLE impressions developed neoplasia. The binary regression model to predict neoplasia had a sensitivity of 83.3%, specificity of 92.5%, and overall accuracy 89.7%. CONCLUSIONS: Presence of malignant criteria and absence of certain inflammatory criteria are more prevalent in patients with neoplasia. Our model, which weights individual imaging components, shows impressive sensitivity and specificity over prior prognostic efforts. Prospective studies will be required to evaluate this model.

Utilization of intraoperative confocal laser endomicroscopy in brain tumor surgery.

Precise identification of tumor margins is of the utmost importance in neuro-oncology. Confocal microscopy is capable of rapid imaging of fresh tissues at cellular resolution and has been miniaturized into handheld probe-based systems suitable for use in the operating room. We aimed to perform a literature review to provide an update on the current status of confocal laser endomicroscopy (CLE) technology for brain tumor surgery. Aside from benchtop confocal microscopes used in ex vivo fashion, there are four CLE systems that have been investigated for potential application in the workflow of brain tumor surgery. Preclinical studies on animal tumor models and clinical studies on human brain tumors have assessed in vivo and ex vivo imaging approaches, suggesting that confocal microscopy holds promise for rapid identification of the characteristic (diagnostic) histological features of tumor and normal brain tissues. However, there are few studies assessing diagnostic accuracy sufficient to provide a definitive determination of the clinical and economical value of CLE in brain tumor surgery. Intraoperative real-time, high-resolution tissue imaging has significant clinical potential in the field of neuro-oncology. CLE is an emerging imaging technology that shows promise for improving brain tumor surgery workflow in in vivo and ex vivo studies. Future clinical studies are necessary to demonstrate clinical and economic benefit of CLE.

Corneal Neurotization for Neurotrophic Keratopathy: Clinical Outcomes and In Vivo Confocal Microscopic and Histopathological Findings.

PURPOSE: To describe the long-term outcomes and in vivo confocal microscopic (IVCM) and histopathological findings after corneal neurotization surgery. METHODS: We included 2 patients who underwent corneal neurotization surgery for severe unilateral neurotrophic keratopathy secondary to cerebellopontine angle meningioma. Corneal sensation was measured using the Cochet-Bonnet esthesiometer (CBE) (0-60 mm). IVCM was performed using the Heidelberg HRT3 Rostock Corneal Module. Histopathological examination was performed on the excised corneoscleral disc of patient 2. RESULTS: In patient 1, corneal sensation improved from 0 mm preoperatively to 60 mm in all 4 quadrants by 2 years postoperatively and was maintained at 5 years postoperatively with identifiable subbasal and stromal corneal nerves on IVCM. In patient 2, corneal sensation improved from 0 mm preoperatively to 10 mm in 3 quadrants (9 months postoperatively) but returned to 0 mm in all quadrants by 2 years postoperatively. IVCM failed to identify any subbasal and stromal corneal nerves. At 5 years postoperatively, evisceration was performed to ameliorate uncontrolled and persistent ocular pain and poor cosmesis. Histopathological examination of the excised corneoscleral disc confirmed the presence of normal-sized, central corneal stromal nerve fascicles but without direct continuity with the transplanted perilimbal nerve bundles. CONCLUSIONS: Our study elucidates the mechanism of corneal neurotization surgery at a cellular level. Although only 1 patient achieved long-term improvement in corneal sensation postoperatively, the findings on IVCM and histopathological examination suggest that partial regeneration/maintenance of corneal nerves after corneal neurotization surgery is likely attributed to the paracrine neurotrophic support, instead of direct sprouting, from the perilimbal transplanted nerve fascicles.

Factors Influencing the Diagnostic Accuracy of Laser-Scanning In Vivo Confocal Microscopy for Acanthamoeba Keratitis.

PURPOSE: To determine the factors that influence the sensitivity and specificity of laser-scanning in vivo confocal microscopy (IVCM) for diagnosing Acanthamoeba keratitis (AK). METHODS: This retrospective, controlled study included 28 eyes of 27 patients with AK and 34 eyes of 34 patients with bacterial keratitis (as the control group). All patients had undergone corneal imaging with a laser-scanning IVCM (Heidelberg Retina Tomograph 3 with the Rostock Cornea Module). The IVCM images were independently evaluated by 2 experienced and 2 inexperienced masked observers. Sensitivity and specificity of IVCM for diagnosing AK and the effects of various clinical and imaging parameters on the sensitivity were then investigated. RESULTS: Overall, IVCM had average sensitivity and specificity of 69.7% ± 2.5% and 97.1% ± 4.2% for experienced observers and 59.0% ± 7.6% and 92.7% ± 10.4% for inexperienced observers, respectively. However, the sensitivity did not show any significant association with the duration of disease, size of ulcer, depth of involvement, culture results, or cyst morphology. Although interobserver agreement was good (κ = 0.60, P < 0.001) for the experienced observers, it was only at a moderate level (κ = 0.48, P < 0.001) for the inexperienced observers. CONCLUSIONS: IVCM has a moderate sensitivity and a high specificity for diagnosis of AK. Although clinical parameters do not affect this diagnostic accuracy, a higher sensitivity is seen when images are interpreted by experienced observers.

In vivo reflectance confocal microscopy in daily practice: Image features correlated to histopathology.

BACKGROUND: In vivo reflectance confocal microscopy (RCM) represents a promising technique for noninvasive visualization of skin lesions. In the clinical daily practice, doctors want to know the relationship between the RCM images and the skin pathological changes. OBJECTIVE: The aim of this study was to identify the basic skin pathological changes under RCM, and use RCM terminology to describe these pathological changes. METHODS: A total of 100 patients were recruited and were evaluated both by RCM and histopathologic examination. Ten healthy volunteers were also recruited as control. RCM examinations were done and biopsies of the lesions at the same site of RCM examination were performed for histopathology analysis. RESULTS: The pathological changes including hyperkeratosis, parakeratosis, acanthosis, papilloma, spongiosis, pustule, vacuolar degeneration, hyperpigmentation, changes of collagen fibers, and vascular changes can be imaged by RCM and corresponded well to their histopathology. RCM failed to find the atypical keratinocytes in two squamous cell carcinoma cases because of the hyperkeratosis and failed to find the vascular changes in one port wine stain cases because of the limitation of detecting depth. CONCLUSION: Features correlating well to histopathology are observed on RCM. RCM can be used as an auxiliary diagnosis tool for the clinical diagnosis.

Immediate Label-Free Ex Vivo Evaluation of Human Brain Tumor Biopsies With Confocal Reflectance Microscopy.

Confocal microscopy utilizing fluorescent dyes is widely gaining use in the clinical setting as a diagnostic tool. Reflectance confocal microscopy is a method of visualizing tissue specimens without fluorescent dyes while relying on the natural refractile properties of cellular and subcellular structures. We prospectively evaluated 76 CNS lesions with confocal reflectance microscopy (CRM) to determine cellularity, architecture, and morphological characteristics. A neuropathologist found that all cases showed similar histopathological features when compared to matched hematoxylin and eosin-stained sections. RNA isolated from 7 tissues following CRM imaging retained high RNA integrity, suggesting that CRM does not alter tissue properties for molecular studies. A neuropathologist and surgical pathologist masked to the imaging results independently evaluated a subset of CRM images. In these evaluations, 100% of images reviewed by the neuropathologist and 95.7% of images reviewed by the surgical pathologist were correctly diagnosed as lesional or nonlesional. Furthermore, 97.9% and 91.5% of cases were correctly diagnosed as tumor or not tumor by the neuropathologist and surgical pathologist, respectively, while 95.8% and 85.1% were identified with the correct diagnosis. Our data indicate that CRM is a useful tool for rapidly screening patient biopsies for diagnostic adequacy, molecular studies, and biobanking.

Characterization of Artifacts Associated With Multicolor Confocal Scanning Laser Ophthalmoscopy.

BACKGROUND AND OBJECTIVE: To characterize the appearance of three types of artifacts observed on multicolor confocal scanning laser ophthalmoscopy (cSLO). PATIENTS AND METHODS: Retrospective review of 159 eyes of 96 consecutive patients from the Duke Eye Center who underwent multicolor cSLO with spectral-domain optical coherence tomography (SD-OCT). Infrared (IR), green, blue, and multicolor reflectance images were evaluated for artifacts with corresponding SD-OCT scans available for reference. RESULTS: Multicolor cSLO artifacts were detected in 23.3% (37 of 159) of eyes and comprised three main patterns: spot, wisp, and net. Only three instances of these artifacts were detected on IR reflectance versus 34, 37, and 35 instances on green, blue, and multicolor reflectance, respectively. Artifacts were observed in 0% of eyes with clear lenses, 27.7% of eyes with cataracts, and in 20.8% of eyes with posterior chamber intraocular lenses. CONCLUSION: Awareness of spot, wisp, and net artifacts when interpreting multicolor cSLO images may facilitate the identification of true retinal pathology. [Ophthalmic Surg Lasers Imaging Retina. 2017;48:810-815.].

A lower dose of fluorescein sodium is more suitable for confocal laser endomicroscopy: a feasibility study.

BACKGROUND AND AIMS: Image quality can be guaranteed with the conventional dosage of fluorescein sodium in probe-based confocal laser endomicroscopy (pCLE). However, yellow discoloration of the skin seriously affects daily life and simultaneously increases the risk of adverse events such as allergic reactions. The aim of this study was to test whether a lower dosage of fluorescein sodium can provide satisfactory image quality and to compare the diagnostic accuracy of gastric intestinal metaplasia (GIM) through a randomized blind controlled trial. METHODS: Consecutive patients were randomly assigned to different doses of fluorescein sodium. Image quality was determined by the endoscopists' subjective assessments and signal-to-noise ratio (SNR) assessment systems. Skin discoloration was tested using a neonatal transcutaneous jaundice detector. In addition, consecutive patients with a known or suspected diagnosis of GIM were examined by pCLE with the lower dose and the traditional dose. RESULTS: Only 0.01 mL/kg dose of 10% fluorescein sodium led to a significant decrease in image quality (P < .05), and a dose of 0.02 mL/kg had the highest SNR value (P < .05). There were no significant differences in skin discoloration between the 0.01 mL/kg and 0.02 mL/kg doses (P = .148) and no statistical difference in the diagnostic accuracy of pCLE for GIM between the 0.02 mL/kg and 0.10 mL/kg doses (P > .05). The kappa values for the correlation between pCLE and histopathology were 0.867 (95% confidence interval, 0.782-0.952) and 0.891 (95% confidence interval, 0.811-0.971). CONCLUSIONS: The 0.02 mL/kg dose of 10% fluorescein sodium seems to be the best dose for pCLE in the upper GI tract, with comparable image quality with the conventional dose and insignificant skin discoloration. This dose is also very efficient for the diagnosis of GIM.

ASGE Technology Committee systematic review and meta-analysis assessing the ASGE Preservation and Incorporation of Valuable Endoscopic Innovations thresholds for adopting real-time imaging-assisted endoscopic targeted biopsy during endoscopic surveillance of Barrett's esophagus.

BACKGROUND AND AIMS: Endoscopic real-time imaging of Barrett's esophagus (BE) with advanced imaging technologies enables targeted biopsies and may eliminate the need for random biopsies to detect dysplasia during endoscopic surveillance of BE. This systematic review and meta-analysis was performed by the American Society for Gastrointestinal Endoscopy (ASGE) Technology Committee to specifically assess whether acceptable performance thresholds outlined by the ASGE Preservation and Incorporation of Valuable Endoscopic Innovations (PIVI) document for clinical adoption of these technologies have been met. METHODS: We conducted meta-analyses calculating the pooled sensitivity, negative predictive value (NPV), and specificity for chromoendoscopy by using acetic acid and methylene blue, electronic chromoendoscopy by using narrow-band imaging, and confocal laser endomicroscopy (CLE) for the detection of dysplasia. Random effects meta-analysis models were used. Statistical heterogeneity was evaluated by means of I(2) statistics. RESULTS: The pooled sensitivity, NPV, and specificity for acetic acid chromoendoscopy were 96.6% (95% confidence interval [CI], 95-98), 98.3% (95% CI, 94.8-99.4), and 84.6% (95% CI, 68.5-93.2), respectively. The pooled sensitivity, NPV, and specificity for electronic chromoendoscopy by using narrow-band imaging were 94.2% (95% CI, 82.6-98.2), 97.5% (95% CI, 95.1-98.7), and 94.4% (95% CI, 80.5-98.6), respectively. The pooled sensitivity, NPV, and specificity for endoscope-based CLE were 90.4% (95% CI, 71.9-97.2), 98.3% (95% CI, 94.2-99.5), and 92.7% (95% CI, 87-96), respectively. CONCLUSIONS: Our meta-analysis indicates that targeted biopsies with acetic acid chromoendoscopy, electronic chromoendoscopy by using narrow-band imaging, and endoscope-based CLE meet the thresholds set by the ASGE PIVI, at least when performed by endoscopists with expertise in advanced imaging techniques. The ASGE Technology Committee therefore endorses using these advanced imaging modalities to guide targeted biopsies for the detection of dysplasia during surveillance of patients with previously nondysplastic BE, thereby replacing the currently used random biopsy protocols.

The learning curve, interobserver, and intraobserver agreement of endoscopic confocal laser endomicroscopy in the assessment of mucosal barrier defects.

BACKGROUND AND AIMS: Confocal laser endomicroscopy can dynamically assess intestinal mucosal barrier defects and increased intestinal permeability (IP). These are functional features that do not have corresponding appearance on histopathology. As such, previous pathology training may not be beneficial in learning these dynamic features. This study aims to evaluate the diagnostic accuracy, learning curve, inter- and intraobserver agreement for identifying features of increased IP in experienced and inexperienced analysts and pathologists. METHODS: A total of 180 endoscopic confocal laser endomicroscopy (Pentax EC-3870FK; Pentax, Tokyo, Japan) images of the terminal ileum, subdivided into 6 sets of 30 were evaluated by 6 experienced analysts, 13 inexperienced analysts, and 2 pathologists, after a 30-minute teaching session. Cell-junction enhancement, fluorescein leak, and cell dropout were used to represent increased IP and were either present or absent in each image. For each image, the diagnostic accuracy, confidence, and quality were assessed. RESULTS: Diagnostic accuracy was significantly higher for experienced analysts compared with inexperienced analysts from the first set (96.7% vs 83.1%, P < .001) to the third set (95% vs 89.7, P = .127). No differences in accuracy were noted between inexperienced analysts and pathologists. Confidence (odds ratio, 8.71; 95% confidence interval, 5.58-13.57) and good image quality (odds ratio, 1.58; 95% confidence interval, 1.22-2.03) were associated with improved interpretation. Interobserver agreement κ values were high and improved with experience (experienced analysts, 0.83; inexperienced analysts, 0.73; and pathologists, 0.62). Intraobserver agreement was >0.86 for experienced observers. CONCLUSION: Features representative of increased IP can be rapidly learned with high inter- and intraobserver agreement. Confidence and image quality were significant predictors of accurate interpretation. Previous pathology training did not have an effect on learning.

Confocal laser endomicroscopy: a new gold standard for the assessment of mucosal healing in ulcerative colitis.

BACKGROUND AND AIM: Endoscopic assessment of mucosal healing in ulcerative colitis (UC) is increasingly accepted as a measure of disease activity, therapeutic goal, and the key prognostic indicator. While regular endoscopy evaluates appearance of the mucosal surface, confocal laser endomicroscopy (CLE) enables in vivo visualization of subepithelial mucosa at 1000× magnification during ongoing endoscopy. Our aims were to determine using CLE whether endoscopically normal appearing colonic mucosa in patients with UC in remission (UC-IR) has fully regenerated mucosal structures, resolved inflammation, and to identify the mechanisms. METHODS: Twelve patients (six controls and six with UC-IR) underwent colonoscopy using CLE and intravenous fluorescein infusion. During colonoscopy, CLE images of colonic mucosa and conventional mucosal biopsies were obtained and evaluated using image-analysis systems. We quantified; (i) regeneration of colonic crypts and blood microvessels; (ii) cyclooxygenase 2 (COX2) expression; (iii) mitochondrial DNA (mtDNA) mutations; (iv) inflammatory infiltration; and (v) vascular permeability (VP). RESULTS: In control subjects, CLE demonstrated normal colonic crypts and microvasculature. COX2 expression was minimal, and < 7% crypts showed mtDNA mutations. Colonic mucosa of UC-IR patients had impaired and distorted crypt regeneration, increased COX2, 69% crypts with mtDNA mutations, persistent inflammation, and abnormal vascular architecture with increased VP (all P < 0.001 vs normal mucosa). CONCLUSIONS: (i) Endoscopically normal appearing colonic mucosa of patients with UC-IR remains abnormal: CLE demonstrates impaired crypt regeneration, persistent inflammation, distinct abnormalities in angioarchitecture and increased vascular permeability; molecular imaging showed increased COX2 and mtDNA mutations; (ii) CLE may serve as a new gold standard for the assessment of mucosal healing in UC.

Screening, early detection, education, and trends for melanoma: current status (2007-2013) and future directions: Part II. Screening, education, and future directions.

New evidence has accumulated over the past several years that supports improved melanoma outcomes associated with both clinician and patient screening. Population-based and workplace studies conducted in Australia and the Unites States, respectively, have shown decreases in the incidence of thick melanoma and overall melanoma mortality, and a year-long statewide screening program in Germany has shown a nearly 50% reduction in mortality 5 years after the screening ended. Current melanoma screening guidelines in the United States are inconsistent among various organizations, and therefore rates of both physician and patient skin examinations are low. As policymaking organizations update national screening recommendations in the United States, the latest research reviewed in part II of this continuing medical education article should be considered to establish the most effective recommendations. Patient and provider education will be necessary to ensure that appropriate patients receive recommended screening.

Utility of confocal laser endomicroscopy in identifying high-grade dysplasia and adenocarcinoma in Barrett's esophagus: a systematic review and meta-analysis.

Confocal laser endomicroscopy (CLE) is a novel endoscopic technique that has emerged as an important tool in the in-vivo visualization and detailed assessment of the mucosal layer and subcellular structures in Barrett's esophagus. Current guidelines recommend four-quadrant random biopsies for identification of high-grade dysplasia (HGD) in Barrett's esophagus. However, random biopsies are associated with sampling error and inconsistent histopathologic interpretation. CLE, by providing targeted biopsies, could decrease the sampling error and increase the yield of detection of HGD/adenocarcinoma [esophageal adenocarcinoma (EAC)]. We carried out a meta-analysis to evaluate the diagnostic accuracy of the CLE-based targeted biopsies in detecting HGD/adenocarcinoma compared with four-quadrant random biopsies. A search using medical subject headings (MeSH) terms and keywords was performed in the MEDLINE and Cochrane review databases and relevant studies were identified. All the studies that compared the diagnostic yield from CLE-based targeted biopsies to detect HGD/adenocarcinoma with a gold standard of histopathology were included and a meta-analysis was carried out to estimate the pooled sensitivity, specificity, and positive and negative likelihood ratios using Meta-Disc software. There were a total of seven studies with 345 patients and 3080 lesions that were finally included in the meta-analysis. All the studies had reported per-lesion analyses; however, only four of the seven studies had data reported on per-patient analyses. 'Per-lesion' analysis for the diagnosis of HGD/adenocarcinoma yielded a pooled sensitivity and specificity of 68% [95% confidence interval (CI) of 64-73%] and 88% (95% CI of 87-89%), respectively. The pooled positive and negative likelihood ratios were 6.56 (95% CI of 3.61-11.90) and 0.24 (95% CI of 0.09-0.63), respectively. Similar numbers were calculated on the basis of 'per-patient' basis, which showed a pooled sensitivity and specificity of 86% (95% CI of 74-96%) and 83% (95% CI of 77-88%), respectively. The pooled positive and negative likelihood ratios were 5.61 (95% CI of 2.00-15.69) and 0.21 (95% CI of 0.08-0.59), respectively. CLE, by providing targeted biopsies, has a good diagnostic accuracy in identifying HGD/EAC; however, the overall prevalence of HGD/EAC in the studies included was much higher than what would be seen in clinical practice and these results should be interpreted with caution. Because of its relatively low sensitivity and negative predictive value, CLE may currently not replace standard biopsy techniques for the diagnosis of HGD/EAC in Barrett's esophagus.

In vivo confocal microscopy in clinical practice: comparison of bedside diagnostic accuracy of a trained physician and distant diagnosis of an expert reader.

BACKGROUND: Reflectance confocal microscopy (RCM) is an imaging tool that allows the visualization of cellular details without biopsy. To our knowledge, RCM sensitivity and specificity has not been studied in a telemedicine setting. OBJECTIVE: We sought to assess RCM diagnostic accuracy in a support teleconsultation setting. METHODS: Between June 2010 and September 2011, 340 lesions were imaged using a confocal scanning microscope. The images were evaluated by 2 readers, one on site, and the other at a distance. RESULTS: A total of 334 cases were included. For each reader the sensitivity was greater than 90% and specificity for each reader was greater than 60%. Both readers had a combined sensitivity of 98.6% and 44% specificity. LIMITATIONS: RCM may be limited in the correct classification of epithelial tumors. CONCLUSIONS: RCM is a tool in the clinical diagnosis of skin lesions, providing a high diagnostic accuracy in teleconsultation use.

Probe-based confocal laser endomicroscopy of the respiratory tract: a data consistency analysis.

BACKGROUND: Probe-based confocal laser endomicroscopy is a novel imaging tool in the field of respiratory medicine. It enables a real-time and bedside qualitative characterization at the level of small airways but due to a relatively small field of view the reliability of quantification remains uncertain. METHODS: Twenty-six lung transplant recipients were subjected to two consecutive alveoloscopic imaging procedures within a median time interval of 90 days to analyze test-retest reliability. Only patients in a stable clinical condition were analyzed. We studied alveolar duct diameter, elastic fiber thickness at the alveolar level, the number of autofluorescent cells at the level of the alveolar space, the diameter of autofluorescent alveolar cells and their autofluorescence intensity. RESULTS: Intraclass correlation coefficients ranged from 0.56 for elastic fiber thickness, 0.62 for alveolar duct diameter, 0.29 for alveolar cell diameter, 0.74 for cellularity to 0.78 for alveolar cell autofluorescence. CONCLUSION: Probe-based confocal laser endomicroscopy enables imaging and quantitative measurements at the level of small airways and alveolar ducts. Test-retest reliability is good for cellularity and autofluorescence quantification but only moderate for morphometric analysis of elastic fibers and alveolar ducts.

A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high-content high-resolution multiparameter image-cytometry.

Dissection of complex molecular-networks in rare cell populations is limited by current technologies that do not allow simultaneous quantification, high-resolution localization, and statistically robust analysis of multiple parameters. We have developed a novel computational platform (Automated Microscopy for Image CytOmetry, A.M.I.CO) for quantitative image-analysis of data from confocal or widefield robotized microscopes. We have applied this image-cytometry technology to the study of checkpoint activation in response to spontaneous DNA damage in nontransformed mammary cells. Cell-cycle profile and active DNA-replication were correlated to (i) Ki67, to monitor proliferation; (ii) phosphorylated histone H2AX (γH2AX) and 53BP1, as markers of DNA-damage response (DDR); and (iii) p53 and p21, as checkpoint-activation markers. Our data suggest the existence of cell-cycle modulated mechanisms involving different functions of γH2AX and 53BP1 in DDR, and of p53 and p21 in checkpoint activation and quiescence regulation during the cell-cycle. Quantitative analysis, event selection, and physical relocalization have been then employed to correlate protein expression at the population level with interactions between molecules, measured with Proximity Ligation Analysis, with unprecedented statistical relevance.