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Probe-based confocal endomicroscopy provides real time videos of autoflourescent elastin structures within the alveoli. With it, multiple changes in the elastin structure due to different diffuse parenchymal lung diseases have previously been described. However, these evaluations have mainly relied on qualitative evaluation by the examiner and manually selected parts post-examination. To develop a fully automatic method for quantifying structural properties of the imaged alveoli elastin and to perform a preliminary assessment of their diagnostic potential. 46 patients underwent probe-based confocal endomicroscopy, of which 38 were divided into 4 groups categorizing different diffuse parenchymal lung diseases. 8 patients were imaged in representative healthy lung areas and used as control group. Alveolar elastin structures were automatically segmented with a trained machine learning algorithm and subsequently evaluated with two methods developed for quantifying the local thickness and structural connectivity. The automatic segmentation algorithm performed generally well and all 4 patient groups showed statistically significant differences with median elastin thickness, standard deviation of thickness and connectivity compared to the control group. Alveoli elastin structures can be quantified based on their structural connectivity and thickness statistics with a fully-automated algorithm and initial results highlight its potential for distinguishing parenchymal lung diseases from normal alveoli.

OBJECTIVE: We aimed to determine the corneal confocal microscopy (CCM) parameter that best identifies diabetic sensorimotor polyneuropathy (DSP) in type 1 diabetes and to describe its performance characteristics. RESEARCH DESIGN AND METHODS: Concurrent with clinical and electrophysiological examination for classification of DSP, CCM was performed on 89 type 1 diabetic and 64 healthy subjects to determine corneal nerve fiber length (CNFL), density, tortuosity, and branch density. Area under the curve (AUC) and optimal thresholds for DSP identification in those with diabetes were determined by receiver operating characteristic (ROC) curve analysis. RESULTS: DSP was present in 33 (37%) subjects. With the exception of tortuosity, CCM parameters were significantly lower in DSP case subjects. In ROC curve analysis, AUC was greatest for CNFL (0.88) compared with fiber density (0.84, P = 0.0001), branch density (0.73, P < 0.0001), and tortuosity (0.55, P < 0.0001). The threshold value that optimized sensitivity and specificity for ruling in DSP was a CNFL of ≤14.0 mm/mm2 (sensitivity 85%, specificity 84%), associated with positive and negative likelihood ratios of 5.3 and 0.18. An alternate approach that used separate threshold values maximized sensitivity (threshold value ≥15.8 mm/mm2, sensitivity 91%, negative likelihood ratio 0.16) and specificity (≤11.5 mm/mm2, specificity 93%, positive likelihood ratio 8.5). CONCLUSIONS: Among CCM parameters, CNFL best discriminated DSP cases from control subjects. A single threshold offers clinically acceptable operating characteristics, although a strategy that uses separate thresholds to respectively rule in and rule out DSP has excellent performance while minimizing unclassified subjects. We hypothesize that values between these thresholds indicate incipient nerve injury that represents those individuals at future neuropathy risk.

AbstractIntroduction: Probe-based confocal laser endomicroscopy (pCLE) is an optical imaging tool which allows live imaging tissues at cellular level. pCLE proved to be able to discriminate malignant from benign pleura during medical thoracoscopy and can identify malignant cells in pleural fluids. Since pleural fluid cytology has limited sensitivity for malignant pleural effusion diagnosis, we wanted do determine the feasibility, the safety, and the yield of mini-invasive close-blinded pleural pCLE. Methods: We conducted a single-center prospective trial. Every patient referred to medical thoracoscopy because of pleural effusion was eligible. Thoracocentesis for pleural fluid analysis was performed first with a Boutin’s needle. Second, the probe was introduced in the pleural space through the Boutin’s needle for mini-invasive pleural pCLE. Finally, the medical thoracoscopy and invasive pleural pCLE were performed. Seven pCLE criteria were prospectively rated to assess the sensitivity and specificity of mini-invasive pleural pCLE for malignant pleural effusion diagnosis. Results: Fifty-two patients were enrolled. Mini-invasive pleural pCLE was feasible and safe. Pathological pleural fluid analysis identified malignant effusion with 42% sensitivity and 100% specificity. For the mini-invasive pleural pCLE, five of 7 criteria proved to be significantly discriminant between malignant and benign pleura. Overall, mini-invasive pleural pCLE had 87% sensitivity for malignant pleural effusion identification. The specificity is 52%, and the positive and negative predictive value were 73% both. Conclusion: Mini-invasive pleural pCLE was safe and feasible and its sensitivity for malignant pleural infiltration diagnosis was much higher than pleural fluid cytology.

Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to ∼3.5 μm/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming.

Corneal confocal microscopy (CCM) derived corneal nerve measures are lower in diabetic sensorimotor polyneuropathy (DSPN). There are, however, methodological challenges in relation to adequate and unbiased sampling of images with objective corneal nerve quantification. Here we compare a new sampling method and adjusted area calculation with established methods of corneal nerve quantification in patients with and without DSPN and healthy controls. CCM images from 26 control subjects and 62 patients with type 1 diabetes with (n = 17) and without (n = 45) DSPN were analyzed. The images were randomly selected and corneal nerve fiber length (CNFL), corneal nerve fiber branch density (CNBD) and corneal nerve fiber density (CNFD) were determined in both a manual and automated manner. The new method generated 8–40% larger corneal nerve parameters compared to the standard procedure ( p  < 0.05). CNFL was significantly reduced using the new method for both manual and automated analysis; whilst CNFD and CNBD were significantly reduced using the automated method in both diabetic groups compared with controls. The new, objective method showed a reduction in corneal nerve parameters in diabetic patients with and without DSPN. We recommend using a randomized sampling method and area-dependent analysis to enable objective unbiased corneal nerve quantification.

CD8 ⁺ T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8 ⁺ T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8 ⁺ T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8 ⁺ T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8 ⁺ T cells and signaling by G protein-coupled receptors, although CD8 ⁺ T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8 ⁺ T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8 ⁺ T cells.

Objective. The aim of this study was to monitor the behavior of interfacial gaps formed under different bonded polymeric restorations before and after thermocycling (TC), using swept-source optical coherence tomography (SS-OCT) and confirming the obtained findings with confocal laser scanning microscopy (CLSM). Materials and Methods. Cylindrical class I cavities were prepared in twenty noncarious human premolar teeth (1.5 mm depth×3.5 mm diameter) and divided randomly into two groups: TS and SN, according to the adhesive system (n=10). In the TS group, one-step self-etch adhesive Clearfil Tri-S Bond Plus (Kuraray Noritake Dental, Japan) was used, followed by composite restoration using Estelite Sigma Quick (Tokuyama Dental, Japan). In the SN group, the cavities were restored with the two-step self-etch/composite silorane-based resin restoration system (3M ESPE, USA). All specimens were restored in bulk filling technique and cured in accordance with the manufacturers’ instructions. Both groups were imaged under SS-OCT after 24 h and recorded as controls. Then, each group was subjected to thermal challenge using the TC machine (5–55°C) and B-scans were recorded at different TC intervals (2600, 5200, and 10000). In order to confirm the SS-OCT findings, additional specimens were prepared, scanned, and sectioned for CLSM observation. Results. B-scans demonstrated white clusters at the tooth-resin interface that corresponded to the gap location on CLSM images. The TS group showed significantly less gap formation than the SN group before and after TC (p<0.001). Conclusions. An optimal composite adaptation can be achieved when the bonded restoration comprises a combination of an adhesive containing 10-MDP monomer and a considerable highly filled composite.

IntroductionDespite many technological advances, the diagnostic yield of bronchoscopic peripheral lung nodule analysis remains limited due to frequent mispositioning. Needle-based confocal laser endomicroscopy (nCLE) enables real-time microscopic feedback on needle positioning, potentially improving the sampling location and diagnostic yield. Previous studies have defined and validated nCLE criteria for malignancy, airway and lung parenchyma. Larger studies demonstrating the effect of nCLE on diagnostic yield are lacking. We aim to investigate if nCLE-imaging integrated with conventional bronchoscopy results in a higher diagnostic yield compared with conventional bronchoscopy without nCLE.Methods and analysisThis is a parallel-group randomised controlled trial. Recruitment is performed at pulmonology outpatient clinics in universities and general hospitals in six different European countries and one hospital in the USA. Consecutive patients with a for malignancy suspected peripheral lung nodule (10–30 mm) with an indication for diagnostic bronchoscopy will be screened, and 208 patients will be included. Web-based randomisation (1:1) between the two procedures will be performed. The primary outcome is diagnostic yield. Secondary outcomes include diagnostic sensitivity for malignancy, needle repositionings, procedure and fluoroscopy duration, and complications. Pathologists will be blinded to procedure type; patients and endoscopists will not.Ethics and disseminationPrimary approval by the Ethics Committee of the Amsterdam University Medical Center. Dissemination involves publication in a peer-reviewed journal.SupportFinancial and material support from Mauna Kea Technologies.Trial registration numberNCT06079970.

Objective We prospectively compared the diagnostic performance of autofluorescence imaging (AFI), magnifying narrow band imaging (mNBI), and probe-based confocal laser endomicroscopy (pCLE) with white light endoscopy (WLE) for the diagnosis of gastric intestinal metaplasia (GIM), using histology as the “gold standard.” Design Chinese >50 years old with history of GIM were prospectively recruited. All subjects underwent WLE, followed by AFI and NBI, and finally pCLE. Patients were randomized to undergo either AFI before NBI or vice versa. In each patient, a minimum of six sites (antrum lesser and greater curve, body lesser and greater curve, incisura, cardia, and any lesion) were each examined by WLE, AFI, NBI, and pCLE. The diagnoses were made real-time. Biopsies for histology were taken from all examined sites. pCLE videos also were reviewed off-site. Analysis was performed per-site. Results A total of 125 sites in 20 patients were examined. For diagnosing GIM, real-time pCLE had better sensitivity (90.9 vs. 37.9 %, p  < 0.001) and accuracy (88.0 vs. 64.8 %, p  < 0.001) compared with WLE. Sensitivity (90.9 vs. 68.2 %, p  = 0.001), specificity (84.7 vs. 69.5 %, p  = 0.042), and accuracy (88 vs. 68.8 %, p  < 0.001) of real-time pCLE were better than AFI. Sensitivity, specificity, and accuracy of real-time pCLE and mNBI for diagnosing GIM were similar. Off-site pCLE had significantly better accuracy for diagnosing GIM compared to WLE, AFI, and mNBI. Off-site pCLE had superior specificity (94.9 vs. 84.7 %, p  = 0.031) and accuracy (95.2 vs. 88.0 %, p  = 0.012) compared with real-time pCLE. Conclusions pCLE was superior to AFI and WLE for diagnosing GIM. Off-site review improved performance of pCLE.

Objective Compare the diagnostic characteristics of intraepidermal nerve fiber density (IENFD) and confocal corneal microscopy (CCM) for distal symmetric polyneuropathy (DSP) and small fiber neuropathy (SFN). Methods Participants with obesity were recruited from bariatric surgery clinics and testing was performed prior to surgery. DSP and SFN were determined using the Toronto consensus definitions of probable neuropathy. IENFD was assessed from 3 mm punch biopsies of the distal leg and proximal thigh. CCM was performed on both eyes with manual and automated counting. The Michigan Neuropathy Screening Instrument questionnaire (MNSIq) was also completed. Diagnostic capability was determined using areas under the receiver operating characteristics curve (AUC) from logistic regression. Results We enrolled 140 participants (mean [standard deviation [SD]] age: 50.3 years [7.1], 77.1% female, BMI: 44.4 kg/m2 [6.7]). In this population, 22.9% had DSP and 14.3% had SFN. Distal leg IENFD had the largest AUC (95% confidence interval) for DSP (0.78, 0.68–0.89) and SFN (0.85, 0.75–0.96). Proximal thigh IENFD (DSP: AUC: 0.59, 0.48–0.69, SFN: AUC: 0.59, 0.46–0.73) and CCM metrics (DSP: AUC range: 0.55–0.60, SFN: AUC range: 0.45–0.62) had poorer diagnostic capability than distal leg IENFD for DSP/SFN (P < 0.05). MNSIq had similar diagnostic capability to distal leg IENFD for both DSP/SFN (DSP: AUC: 0.76, 0.68–0.85, SFN: AUC: 0.81, 0.73–0.88). More participants (52%) preferred skin biopsies to CCM. Interpretation Distal leg IENFD was the best quantitative measure of DSP/SFN. CCM had poor diagnostic characteristics and fewer patients preferred this test to IENFD. The MNSIq had similar diagnostic characteristics to distal leg IENFD, indicating its value as a diagnostic tool in the clinical setting. Clinical Trial Registration clinicaltrials.gov: NCT03617185.