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.

3D test sample for the calibration and quality control of stimulated emission depletion (STED) and confocal microscopes.

Multiple samples are required to monitor and optimize the quality and reliability of quantitative measurements of stimulated emission depletion (STED) and confocal microscopes. Here, we present a single sample to calibrate these microscopes, align their laser beams and measure their point spread function (PSF) in 3D. The sample is composed of a refractive index matched colloidal crystal of silica beads with fluorescent and gold cores. The microscopes can be calibrated in three dimensions using the periodicity of the crystal; the alignment of the laser beams can be checked using the reflection of the gold cores; and the PSF can be measured at multiple positions and depths using the fluorescent cores. It is demonstrated how this sample can be used to visualize and improve the quality of STED and confocal microscopy images. The sample is adjustable to meet the requirements of different NA objectives and microscopy techniques and additionally can be used to evaluate refractive index mismatches as a function of depth quantitatively.

A plea for standardization of confocal microscopy and optical coherence tomography parameters to evaluate physiological and para-physiological skin conditions in cosmetic science.

Non-invasive reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) have been extended to the dermo-cosmetic field, for skin pathophysiology understanding and therapeutics monitoring. However, standardized methodology and parameters to interpret structures and changes in these settings are still lacking. Present study aimed to propose a validated standard methodology and a list of defined parameters for objective non-pathological skin assessments in the cosmetically sensitive cheekbone area of the face. OCT and RCM quantitative, semi-quantitative and qualitative features were considered for assessments. Validation process included 50 sets of images divided into two age groups. Inter-rater reliability was explored to assess the influence of the proposed methodology. Quantitative OCT parameters of "epidermal thickness," "density and attenuation coefficients" and "vascular density" were considered and calculated. Severity scales were developed for semi-quantitative OCT features of "disruption of collagen" and "vascular asset," while extent scales were produced for semi-quantitative RCM "irregular honeycomb," "mottled pigmentation" and "polycyclic papillary contours." Qualitative assessment was obtained for RCM type of collagen, and comparison between age groups was performed for all features considered. Severity visual scales assistance proved excellent inter-rater agreement across all semi-quantitative and qualitative domains. The assistance of shareable software systems allows for objective OCT quantitative parameters measurement. The use of standard reference scales, within a defined assessment methodology, offers high inter-rater reliability and thus reproducibility for semi-quantitative and qualitative OCT and RCM parameters. Taken together, our results may represent a starting point for a standardized application of RCM and OCT in dermo-cosmetic research and practice.

Corneal confocal microscopy differentiates inflammatory from diabetic neuropathy.

BACKGROUND: Immune-mediated neuropathies, such as chronic inflammatory demyelinating polyneuropathy (CIDP) are treatable neuropathies. Among individuals with diabetic neuropathy, it remains a challenge to identify those individuals who develop CIDP. Corneal confocal microscopy (CCM) has been shown to detect corneal nerve fiber loss and cellular infiltrates in the sub-basal layer of the cornea. The objective of the study was to determine whether CCM can distinguish diabetic neuropathy from CIDP and whether CCM can detect CIDP in persons with coexisting diabetes. METHODS: In this multicenter, case-control study, participants with CIDP (n = 55) with (n = 10) and without (n = 45) diabetes; participants with diabetes (n = 58) with (n = 28) and without (n = 30) diabetic neuropathy, and healthy controls (n = 58) underwent CCM. Corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal nerve branch density (CNBD), and dendritic and non-dendritic cell density, with or without nerve fiber contact were quantified. RESULTS: Dendritic cell density in proximity to corneal nerve fibers was significantly higher in participants with CIDP with and without diabetes compared to participants with diabetic neuropathy and controls. CNFD, CNFL, and CNBD were equally reduced in participants with CIDP, diabetic neuropathy, and CIDP with diabetes. CONCLUSIONS: An increase in dendritic cell density identifies persons with CIDP. CCM may, therefore, be useful to differentiate inflammatory from non-inflammatory diabetic neuropathy.

Comparison of true-colour wide-field confocal scanner imaging with standard fundus photography for diabetic retinopathy screening.

BACKGROUND: Screening of diabetic retinopathy (DR) reduces blindness by early identification of retinopathy. This study compares DR grades derived from a two-field imaging protocol from two imaging platforms, one providing a single 60-degree horizontal field of view (FOV) and the other, a standard 45-degree FOV. METHODS: Cross-sectional study which included 1257 diabetic patients aged ≥18 years attending their DR screening visit in the English National Diabetic Eye Screening Programme (NDESP). Patients with maculopathy (M1), preproliferative (R2) or proliferative DR (R3) were referred to an ophthalmologist. Patients with ungradable images (U) are examined in a slit-lamp biomicroscopy clinic. Image acquisition under mydriasis of two images per eye was carried out with the EIDON and with standard fundus cameras. Evaluation was performed by masked graders. RESULTS: Agreement after consensus with kappa statistic was 0.89 (quadratic weights (95% CI 0.87 to 0.92)) for NDESP severity grade, 0.88 (quadratic weights (95% CI 0.82 to 0.94)) for referable disease and 0.92 (linear weights (95% CI 0.88 to 0.95)) for maculopathy. The EIDON detected clinically relevant DR features outside the 45-degree fields in two patients (0.16%): one with intraretinal microvascular abnormalities (IRMAs) and one with neovascularisation. In eight patients (0.64%), the EIDON allowed DR feature visualisation inside the 45-degree fields that were not identified in the NDESP images: three patients (0.24%) with IRMA and five patients (0.40%) with maculopathy. The rates of ungradable encounters were 12 (0.95%) and 13 (1.03%) with the EIDON and NDESP images, respectively. CONCLUSION: The EIDON identifies a small number of additional patients with referable disease which are not detected with standard imaging. This is due to the EIDON finding disease outside the standard FOV and greater clarity finding disease within the standard FOV.

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.

Utilizing Machine Learning for Image Quality Assessment for Reflectance Confocal Microscopy.

In vivo reflectance confocal microscopy (RCM) enables clinicians to examine lesions' morphological and cytological information in epidermal and dermal layers while reducing the need for biopsies. As RCM is being adopted more widely, the workflow is expanding from real-time diagnosis at the bedside to include a capture, store, and forward model with image interpretation and diagnosis occurring offsite, similar to radiology. As the patient may no longer be present at the time of image interpretation, quality assurance is key during image acquisition. Herein, we introduce a quality assurance process by means of automatically quantifying diagnostically uninformative areas within the lesional area by using RCM and coregistered dermoscopy images together. We trained and validated a pixel-level segmentation model on 117 RCM mosaics collected by international collaborators. The model delineates diagnostically uninformative areas with 82% sensitivity and 93% specificity. We further tested the model on a separate set of 372 coregistered RCM-dermoscopic image pairs and illustrate how the results of the RCM-only model can be improved via a multimodal (RCM + dermoscopy) approach, which can help quantify the uninformative regions within the lesional area. Our data suggest that machine learning-based automatic quantification offers a feasible objective quality control measure for RCM imaging.

Trueness and Precision of Two Intraoral Scanners: A Comparative In Vitro Study.

The aim of this study was to evaluate the accuracy of two intraoral scanners used in the dental office. A molar fixed in a typodont was prepared for a ceramic onlay. The preparation was scanned using a high-resolution scanner (reference scanner) and saved as stereolithography (STL) format. The prepared resin molar was scanned again using the intraoral scanners, and all the scans were saved as well in STL format. All STL files were compared using metrology software (Geomagic Control X). Overlapping the meshes allowed the assessment of the scans in terms of trueness and precision. Based on the results of this study, the differences of trueness and precision between the intraoral scanners were minimal.

Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy.

Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol.

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.

Implementation of a Quality Index for Improvement of Quantification of Corneal Nerves in Corneal Confocal Microcopy Images: A Multicenter Study.

PURPOSE: Corneal confocal microscopy (CCM) is an imaging method to detect loss of nerve fibers in the cornea. The impact of image quality on the CCM parameters has not been investigated. We developed a quality index (QI) with 3 stages for CCM images and compared the influence of the image quality on the quantification of corneal nerve parameters using 2 modes of analysis in healthy volunteers and patients with known peripheral neuropathy. METHODS: Images of 75 participants were a posteriori analyzed, including 25 each in 3 image quality groups (QI 1-QI 3). Corneal nerve fiber length (CNFL) was analyzed using automated and semiautomated software, and corneal nerve fiber density and corneal nerve branch density were quantified using automated image analysis. Three masked raters assessed CCM image quality (QI) independently and categorized images into groups QI 1-QI 3. In addition, statistical analysis was used to compare interrater reliability. Analysis of variance was used for analysis between the groups. Interrater reliability analysis between the image ratings was performed by calculating Fleiss' kappa and its 95% confidence interval. RESULTS: CNFL, corneal nerve fiber density, and corneal nerve branch density increased significantly with QI (P < 0.001, all post hoc tests P < 0.05). CNFL was higher using semiautomated compared with automated nerve analysis, independent of QI. Fleiss kappa coefficient for interrater reliability of QI was 0.72. CONCLUSIONS: The quantification of corneal nerve parameters depends on image quality, and poorer quality images are associated with lower values for corneal nerve parameters. We propose the QI as a tool to reduce variability in quantification of corneal nerve parameters.

How transparent film applied on dermatologic imaging devices in order to prevent infections affects image quality?

BACKGROUND: In the clinical practice, transparent films are used as sterile interfaces in in vivo dermatologic imaging in order to prevent the transmissions of infections. However, in our experience, the use of a transparent film can alter skin images. Our study aimed to compare the optical quality of a series of different plastic films used as interfaces in order to understand if some might be more suitable for imaging. MATERIALS AND METHODS: We tested the optical properties of 11 different protective transparent films that are marketed in France with a transparency meter and a spectrophotometer. RESULTS: Transmission, minimal diffusion, amount of gray, and contrast were obtained for each transparent film. Transmission ranged from 93.24% to 96.88% (mean 95.36; standard deviation SD 1.02), minimal diffusion from 88.28% to 123.87% (mean 101.04; standard deviation SD 10.02) and contrast from 11.01 to 15.88 (mean 13.93 and SD 1.3). For some films, the transmission was lower at lower wavelengths. CONCLUSION: All tested films had excellent optical properties. However, some of them had better optical qualities and seemed more suitable for their use in dermatologic imaging.

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement. In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

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.

Impact of Vial Washing and Depyrogenation on Surface Properties and Delamination Risk of Glass Vials.

PURPOSE: The proper understanding of glass delamination is important to glass manufacturers, pharmaceutical companies, and health authorities to mitigate the occurrence of glass flakes from the vial when in contact with specific drug product solutions. The surface of glass vials is altered during glass cane- and vial forming processes and is exposed to different stress conditions during drug product processing before coming in contact with the drug product solution. In this study, the impact of vial washing and depyrogenation including an evaluation of various residual water volumes on surface properties of glass vials was investigated for a defined set of vials. METHODS: 3D laser scanning microscopy was established as a new method for topographic analysis of curved surfaces of glass vials operating in high-throughput mode. A subset of vials was subsequently exposed to delamination stress testing and both the stressed solution and inner vial surface were analyzed by a panel of conventional and advanced analytical techniques including 3D laser scanning microscopy. RESULTS: The data showed that vial washing and depyrogenation strongly influenced surface properties, in particular those of uncoated vials. Surface characteristics such as pits increased depending on the process conditions, which especially applies to Expansion 33 vials. Even low residual water volumes of 50 µL after vial washing were sufficient to change the surface properties of the glass and weaken the surface in those positions prone to glass delamination. An increase in pits was related to a greater risk for glass delamination. CONCLUSIONS: Vial processing conditions need to be assessed when aiming at minimizing the glass delamination risk during parenteral product storage.

Corneal confocal microscopy detects severe small fiber neuropathy in diabetic patients with Charcot neuroarthropathy.

AIMS/INTRODUCTION: The aim of the present study was to identify the extent of small fiber neuropathy in diabetic patients with Charcot neuroarthropathy (CN). MATERIALS AND METHODS: A total of 20 patients with CN were compared with 20 age- and diabetes duration-matched patients with type 2 diabetes and 20 age-matched control participants. All patients underwent corneal confocal microscopy with quantification of corneal nerve morphology and assessment for vibration perception threshold, and a subset of patients with CN underwent assessment of sudomotor function and neuropathic pain. RESULTS: In patients with CN compared with type 2 diabetes patients and control participants, there was a significant reduction in corneal nerve fiber density (14.94 ± 8.23 vs 23.86 ± 7.71, P = 0.004 vs 34.84 ± 9.13, P < 0.001), corneal nerve branch density (18.61 ± 16.7 vs 41.62 ± 22.67, P = 0.032 vs 76.47 ± 38.44, P < 0.001) and corneal nerve fiber length (8.40 ± 4.83 vs 14.87 ± 4.76, P = 0.001 vs 21.24 ± 6.48, P < 0.001), electrochemical skin conductance on the feet (20.57 ± 13.99 vs 61.50 ± 22.26, P < 0.001 vs 76.23 ± 12.01, P < 0.001) and hands (30.86 ± 18.10 vs 61.13 ± 19.14, P = 0.001 vs 68.31 ± 11.96, P < 0.001), and a significant increase in the vibration perception threshold in the feet (38.46 ± 15.10 vs 14.15 ± 10.25, P < 0.001 vs 7.75 ± 4.01, P < 0.001). CONCLUSIONS: Patients with diabetes and CN have severe large and particularly small fiber neuropathy.

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.

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.