AIM: To evaluate the corneal cell morphology of new keratoconus patients wearing two different types of rigid gas-permeable(RGP) contact lenses for 1y.METHODS: Thirty nine eyes of 39 new keratoconus patients were ...AIM: To evaluate the corneal cell morphology of new keratoconus patients wearing two different types of rigid gas-permeable(RGP) contact lenses for 1y.METHODS: Thirty nine eyes of 39 new keratoconus patients were selected and randomly fitted with two types of RGP contact lenses.Group 1 had 21 eyes with regular rigid gas-permeable(RRGP) contact lens and rest 18 eyes were in group 2 with specially designed rigid gas-permeable(SRGP) contact lens.Corneal cell morphology was evaluated using a slit scanning confocal microscope at no-lens wear and after 1y of contact lens wearing.RESULTS: After 1y of contact lens wearing in group 1,the mean anterior and posterior stromal keratocyte density were significantly less(P=0.006 and P=0.001,respectively) compared to no-lens wear.The mean cell area of anterior and posterior stromal keratocyte were also significantly different(P=0.005 and P=0.001) from no-lens wear.The anterior and posterior stromal haze increased by 18.74% and 23.81%,respectively after 1y of contact lens wearing.Whereas in group 2,statistically significant changes were observed only in cell density & area of anterior stroma(P=0.001 and P=0.001,respectively) after 1y.While,level of anterior and posterior stromal haze increased by 16.67% and 11.11% after 1y of contact lens wearing.Polymegathism and pleomorphism also increased after 1y of contact lens wearing in both the contact lens groups.CONCLUSION: Confocal microscopy observation shows the significant alterations in corneal cell morphology of keratoconic corneas wearing contact lenses especially in group 1.The type of contact lens must be carefully selected to minimize changes in corneal cell morphology.展开更多
AIM: To describe the clinical features and microstructural characteristics assessed by in vivo confocal microscopy(IVCM) in patients with ocular cicatricial pemphigoid(OCP).· METHODS: A descriptive, uncontr...AIM: To describe the clinical features and microstructural characteristics assessed by in vivo confocal microscopy(IVCM) in patients with ocular cicatricial pemphigoid(OCP).· METHODS: A descriptive, uncontrolled case series study. Patients diagnosed with OCP were examined by clinical history, slit-lamp biomicroscopy features and IVCM images. The results of direct immunofluorescence(DIF) biopsies and indirect immunofluorescence(IIF) were also recorded. Local and systemic immunosuppressive therapy were administered and adjusted according to response.·RESULTS: A total of 12 consecutive OCP patients(7male, 5 female; mean age 60.42 ±10.39y) were recruited.All patients exhibited bilateral progressive conjunctival scarring and recurrent chronic conjunctivitis was the most frequent clinical pattern. The mean duration of symptoms prior to diagnosis of OCP was 2.95 ±2.85y(range: 5mo to 10y). The Foster classification varied from stage I to IV and 20 eyes(83%) were within or greater than Foster stage Ⅲ on presentation. Two of the 12patients(17%) demonstrated positive DIF; 3 of the 12(25%) patients reported positive IIF. The mean duration of the follow-up period was 20.17 ±11.88mo(range: 6 to48mo). IVCM showed variable degrees of abnormality in the conjuctiva-cornea and conjuctival scarring was detected in all the involved eyes. Corneal stromal cell activation and dendritic cell infiltration presented asocular surface inflammation, ocular surface keratinization along with the destroyed Vogt palisades was noted in eyes with potential limbal stem cell deficiency. After treatment, remission of ocular surface inflammation was achieved in all the patients, 18 eyes(75%) remained stable, 6 eyes(25%) had recurrent conjunctivitis and cicatrization in 2 eyes(8%) was progressing.· CONCLUSION: As an autoimmune disease, OCP manifests as variable degrees of clinical and laboratory abnormalities with both local and systemic immunosuppressive treatment playing important roles in disease therapy. IVCM can be as a valuable non-invasive technique to assess ocular surface changes in a cellular level with a potential value for providing diagnostic evidence and monitoring therapeutic effects during follow-up.展开更多
Objective:Novel optical imaging modalities are under development with the goal of obtaining an“optical biopsy”to efficiently provide pathologic details.One such modality is confocal microscopy which allows in situ v...Objective:Novel optical imaging modalities are under development with the goal of obtaining an“optical biopsy”to efficiently provide pathologic details.One such modality is confocal microscopy which allows in situ visualization of cells within a layer of tissue and imaging of cellular-level structures.The goal of this study is to validate the ability of confocal microscopy to quickly and accurately differentiate between normal renal tissue and cancer.Methods:Specimens were obtained from patients who underwent robotic partial nephrectomy for renal mass.Samples of suspected normal and tumor tissue were extracted from the excised portion of the kidney and stained with acridine orange.The stained samples were imaged on a Nikon E600 C1 Confocal Microscope.The samples were then submitted for hematoxylin and eosin processing and read by an expert pathologist to provide a gold-standard diagnosis that can later be compared to the confocal images.Results:This study included 11 patients,17 tissue samples,and 118 confocal images.Of the 17 tissue samples,10 had a gold-standard diagnosis of cancer and seven were benign.Of 118 confocal images,66 had a gold-standard diagnosis of cancer and 52 were benign.Six confocal images were used as a training set to train eight observers.The observers were asked to rate the test images on a six point scale and the results were analyzed using a web based receiver operating characteristic curve calculator.The average accuracy,sensitivity,specificity,and area under the empirical receiver operating characteristic curve for this study were 91%,98%,81%,and 0.94 respectively.Conclusion:This preliminary study suggest that confocal microscopy can be used to distinguish cancer from normal tissue with high sensitivity and specificity.The observers in this study were trained quickly and on only six images.We expect even higher performance as observers become more familiar with the confocal images.展开更多
Objective To study the effect of ginsenoside Rb1 and total saponin of dipsacus asper on intracellular free calcium concentration mediated by β amyloid protein.So as to lay a foundation for developing effective Chines...Objective To study the effect of ginsenoside Rb1 and total saponin of dipsacus asper on intracellular free calcium concentration mediated by β amyloid protein.So as to lay a foundation for developing effective Chinese traditional medicine to treat Alzheimer’s disease.Methods The technique of laser scanning confocal microscopy combining primary cultured neurons was adopted to quantitatively analyze the change of [Ca 2+ ] i.Results The [Ca 2+ ] i of primary cultured hippocampal neurons was nmol·L -1 on basal levels.Control group showed obvious change of calcium vibration,[Ca 2+ ] i was elevated to nmol·L -1 .The peak of [Ca 2+ ] i of Rb1 group reached nmol·L -1 and was lower than that of control group .The tSDA group displayed distinct change of calcium vibration too,and [Ca 2+ ] i reached nmol·L -1 .There was a significant difference in [Ca 2+ ] i between control and tSDA group .Conclusion The research indicated that one of mechanisms by which Rb1 and tSDA protected the neurons was to maintain the balance of [Ca 2+ ] i.展开更多
Confocal microscopy is a method which has been increasingly used over the last decade in the study of the anterior ocular surface. The method allows testing and <em>in vivo</em> high resolution imaging of ...Confocal microscopy is a method which has been increasingly used over the last decade in the study of the anterior ocular surface. The method allows testing and <em>in vivo</em> high resolution imaging of the structures of the anterior eye segment, at a cellular level, which is close to the histological examination of tissues. The data provided by this method allow for a better understanding of both the functional and pathological processes occurring in the anterior ocular surface not only for scientific purposes but also in clinical practice. The aim of the present work is to summarize the current knowledge and applications of confocal microscopy of the anterior ocular surface.展开更多
Background:To compare the efficacy of Ex-PRESS implantation versus trabeculectomy combined with phacoemulsification.Methods:A retrospective 12-month study on patients with coincident primary open-angle glaucoma(POAG)a...Background:To compare the efficacy of Ex-PRESS implantation versus trabeculectomy combined with phacoemulsification.Methods:A retrospective 12-month study on patients with coincident primary open-angle glaucoma(POAG)and cataract.The patients underwent combined phacoemulsification and Ex-PRESS implant(Phaco-ExPRESS,n=35)or phacotrabeculectomy(Phaco-Trab,n=35).The morphological structures of the filtering bleb were examined by slitlamp,anterior segment optical coherence tomography(AS-OCT)and in vivo confocal microscopy(IVCM).Complete success was defined as postoperative intraocular pressure(IOP)<18 mmHg without the use of anti-glaucoma medication.Qualified success was defined as postoperative IOP<18 mmHg with or without anti-glaucoma medications.The data were collected preoperatively and postoperatively at 2 weeks,1 month,3 months,6 months,and 12 months.Results:No significant difference in the variables such as age,IOP and perimetry was found between the groups of Phaco-ExPRESS and Phaco-Trab.At the one-year postoperative visit for filtering blebs,Phaco-ExPRESS increased the mean area of epithelial microcysts significantly from 0.10±0.05 to 0.20±0.09μm^(2)perμm^(2),while Phaco-Trab decreased the mean area significantly from 0.08±0.04 to 0.04±0.06μm^(2)perμm^(2).Notably,the hyperreflective dots detected by IVCM decreased by 84.9%in Phaco-ExPRESS but increased by 36.3%in Phaco-Trab.The hyperreflective dots were further identified as neutrophil-and monocyte-like cells.The number of these cells were negatively correlated with the microcysts area(r=−0.7,P<0.01)but positively associated with the grade of connective tissue(r=0.5,P<0.01).By creating different microstructural changes in the filtering blebs,Phaco-ExPRESS produced a higher complete success rate(84.9%vs.41.2%,P<0.01)and significant decrease in the number of anti-glaucoma medications(P<0.01)when compared with those in Phaco-Trab.However,the qualified success showed no significant difference between the two groups(100.0%vs.91.2%,P=0.24).Conclusions:At the one-year follow-up,Phaco-ExPRESS generated better filtering bleb with larger area of microcysts,looser connective tissues,and less inflammation than that of Phaco-Trab,providing adequate IOP control and less IOP-lowering medications.These findings indicate that Phaco-ExPRESS could be more preferred than Phaco-Trab for the treatment of patients with coincident POAG and cataract.展开更多
AIM:To evaluate the effects of antiglaucoma eye drops on corneal nerves by in vivo confocal microscopy(IVCM).METHODS:This study comprised 79 patients diagnosed with glaucoma and 16 healthy control individuals.Among th...AIM:To evaluate the effects of antiglaucoma eye drops on corneal nerves by in vivo confocal microscopy(IVCM).METHODS:This study comprised 79 patients diagnosed with glaucoma and 16 healthy control individuals.Among the glaucoma patients,54 were treated with medication,while 25 remained untreated.Central corneal images were evaluated by IVCM,and then ACCMetrics was used to calculate the following parameters:corneal nerve fiber density(CNFD),branch density(CNBD),fiber length(CNFL),total branch density(CTBD),fiber area(CNFA),fiber width(CNFW),and fractal dimension(CNFrD).The correlation between IVCM parameters and drugs was evaluated using non-parametric measurements of Spearman’s rank correlation coefficient.RESULTS:The CNFD was reduced in glaucoma groups compared to healthy subjects(P<0.01).Patients using anti-glaucoma medications exhibited poorer confocal parameters compared to untreated patients.As the number of medications and usage count increased,CNFD,CNBD,CNFL,CTBD,CNFA,and CNFrD experienced a decline,while CNFW increased(all P<0.01).For the brinzolamide-therapy group,there was a significant decrease in CNFD and CNFL compared to the other monotherapy groups(P<0.001).In the absence of medication,CNFD in males was lower than that in females(P<0.05).Among patients under medication therapy,CNFD remained consistent between males and females.CONCLUSION:Antiglaucoma eye drops affect the microstructure of corneal nerves.IVCM and ACCMetrics are useful tools that could be used to evaluate the corneal nerve changes.展开更多
Conventional microscopes designed for submicron resolution in biological research are hindered by a limited field of view,typically around 1 mm.This restriction poses a challenge when attempting to simultaneously anal...Conventional microscopes designed for submicron resolution in biological research are hindered by a limited field of view,typically around 1 mm.This restriction poses a challenge when attempting to simultaneously analyze various parts of a sample,such as different brain areas.In addition,conventional objective lenses struggle to perform consistently across the required range of wavelengths for brain imaging in vivo.Here we present a novel mesoscopic objective lens with an impressive field of view of 8 mm,a numerical aperture of 0.5,and a working wavelength range from 400 to 1000 nm.We achieved a resolution of 0.74μm in fluorescent beads imaging.The versatility of this lens was further demonstrated through high-quality images of mouse brain and kidney sections in a wide-field imaging system,a confocal laser scanning system,and a two-photon imaging system.This mesoscopic objective lens holds immense promise for advancing multi-wavelength imaging of large fields of view at high resolution.展开更多
Background:Dry eye patients suffer from all kinds of symptoms.Sometimes,the clinical signs evaluation does not disclose any obvious difference in routine examination;in vivo confocal microscopy (IVCM) is a powerful...Background:Dry eye patients suffer from all kinds of symptoms.Sometimes,the clinical signs evaluation does not disclose any obvious difference in routine examination;in vivo confocal microscopy (IVCM) is a powerful tool for ocular surface disease.This study aimed to clarify meibomian gland (MG) alterations in dry eye patients with different symptoms and to compare the findings using IVCM.Methods:A total of sixty patients were recruited,all subjected to Ocular Surface Disease Index (OSDI) and Salisbury Eye Evaluation Questionnaire (SEEQ),and questionnaires for the assessment of dry eye symptoms before clinical sign examinations were given to the patients.Finally,IVCM was applied to observe MG's structure.Statistical analysis was performed using the t-test,Mann-Whitney U-test and Spearman correlation analysis.The differences were statistically significant when P 〈 0.05.Results:In the severe symptom group,OSDI and SEEQ scores were significantly higher (P 〈 0.05) compared with the mild symptoms group.All other clinical sign examinations had no statistical difference in the two groups (P 〉 0.05).However,all the IVCM-observed data showed that patients with severe symptoms had more significant fibrosis in MG (acinar unit area 691.87 ± 182.01 μm^2 for the severe,992.17 ± 170.84 μm^2 for the mild;P 〈 0.05) and severer decrease in the size of MG acinar units than those observed in patients with mild symptoms (MG acinar unit density [MGAUD] 70.08 ± 18.78 glands/mm2,MG acinar unit longest diameter [MGALD] 51.50 ± 15.51 μm,MG acinar unit shortest diameter [MGASD] 20.30 ± 11.85 μm for the severe,MGAUD 89.53 ± 39.88 glands/mm2,MGALD 81.57 ± 21.14 μm,MGASD 42.37 ± 14.55 μm for the mild;P 〈 0.05).Dry eye symptoms were negatively correlated with MG confocal microscopic parameters and positively correlated with conjunctival inflammatory cells and Langerhans cells (P 〈 0.05).Conclusions:IVCM application provides a strong support to differentiate dry eye patients with different symptoms:meibomian gland dysfunction (MGD) plays a pivotal role in dry eye aggravation,and using IVCM to observe MG fibrosis,changes in size and density of MG as well as status of inflammation cells can help not only correctly diagnose the type and severity of dry eye,but also possibly prognosticate in routine eye examination in the occurrence of MGD.展开更多
Objective To examine the effects of melatonin on the dynamic changes in the concentration of intracellular free Ca 2+ ([Ca 2+ ]i) in single intact cultured cortical neurons isolated from fetal rats, in order...Objective To examine the effects of melatonin on the dynamic changes in the concentration of intracellular free Ca 2+ ([Ca 2+ ]i) in single intact cultured cortical neurons isolated from fetal rats, in order to explore the possible antiaging mechanisms of melatonin (MT) Methods Using the highly fluorescent Ca 2+ sensitive indicator Fluo 3/AM, cortical neurons cultured in a 35?mm Tissue Culture Dish were in incubated for 45?min at room temperature with 5?μmol/L Fluo 3/AM, resulting in proper intracellular dye concentration to provide adequate signal strength for detection and excellent Laser Scanning Confocal Microscopy (LSCM) imaging of [Ca 2+ ]i while not disturbing normal intracellular physiology The changes in fluorescent intensity were monitored by LSCM Results Bay K8644 (10 6 ?mol/L), KCl (20 ?mmol/L), sodium L glutamate (Glu, 50?μmol/L) caused a rapid increase of [Ca 2+ ]i in cortical neurons, and this increase could be significantly attenuated by 10 6 and 10 7 mol/L MT Conclusions MT could antagonize the extracellular Ca 2+ influx, reduce Ca 2+ overload, and have a protective effect on neurons This may be one of the important antiaging mechanisms of MT展开更多
Parkinson's disease(PD)is a chronic,progressive neurodegenerative disease affecting about 2%-3% of population above the age of 65.In recent years,Parkinson's research has mainly focused on motor and non-motor ...Parkinson's disease(PD)is a chronic,progressive neurodegenerative disease affecting about 2%-3% of population above the age of 65.In recent years,Parkinson's research has mainly focused on motor and non-motor symptoms while there are limited studies on neurodegeneration which is associated with balance problems and increased incidence of falls.Corneal confocal microscopy(CCM)is a real-time,non-invasive,in vivo ophthalmic imaging technique for quantifying nerve damage in peripheral neuropathies and central neurodegenerative disorders.CCM has shown significantly lower corneal nerve fiber density(CNFD)in patients with PD compared to healthy controls.Reduced CNFD is associated with decreased intraepidermal nerve fiber density in PD.This review provides an overview of the ability of CCM to detect nerve damage associated with PD.展开更多
BACKGROUND Currently,there is insufficient data about the accuracy in the diagnosing of pancreatic cystic lesions(PCLs),especially with novel endoscopic techniques such as with direct intracystic micro-forceps biopsy(...BACKGROUND Currently,there is insufficient data about the accuracy in the diagnosing of pancreatic cystic lesions(PCLs),especially with novel endoscopic techniques such as with direct intracystic micro-forceps biopsy(mFB)and needle-based confocal laser-endomicroscopy(nCLE).AIM To compare the accuracy of endoscopic ultrasound(EUS)and associated techniques for the detection of potentially malignant PCLs:EUS-guided fine needle aspiration(EUS-FNA),contrast-enhanced EUS(CE-EUS),EUS-guided fiberoptic probe cystoscopy(cystoscopy),mFB,and nCLE.METHODS This was a single-center,retrospective study.We identified patients who had undergone EUS,with or without additional diagnostic techniques,and had been diagnosed with PCLs.We determined agreement among malignancy after 24-mo follow-up findings with detection of potentially malignant PCLs via the EUSguided techniques and/or EUS-guided biopsy when available(EUS malignancy detection).RESULTS A total of 129 patients were included, with EUS performed alone in 47/129. In 82/129 patients,EUS procedures were performed with additional EUS-FNA (21/82), CE-EUS (20/82), cystoscopy(27/82), mFB (36/82), nCLE (44/82). Agreement between EUS malignancy detection and the 24-mo follow-up findings was higher when associated with additional diagnostic techniques thanEUS alone [62/82 (75.6%) vs 8/47 (17%);OR 4.35, 95%CI: 2.70-7.37;P < 0.001]. The highestmalignancy detection accuracy was reached when nCLE and direct intracystic mFB were bothperformed, with a sensitivity, specificity, positive predictive value, negative predictive value andobserved agreement of 100%, 89.4%, 77.8%, 100% and 92.3%, respectively (P < 0.001 comparedwith EUS-alone).CONCLUSIONThe combined use of EUS-guided mFB and nCLE improves detection of potentially malignantPCLs compared with EUS-alone, EUS-FNA, CE-EUS or cystoscopy.展开更多
Purpose:Assessing the quality of the ocular surface by in vivo scanning laser confocal microscopy(IVCM)in primary open angle glaucoma(POAG)patients treated by Xen 45 Gel Stent,medical therapy and trabeculectomy.Method...Purpose:Assessing the quality of the ocular surface by in vivo scanning laser confocal microscopy(IVCM)in primary open angle glaucoma(POAG)patients treated by Xen 45 Gel Stent,medical therapy and trabeculectomy.Methods:Retrospective,single-center,single-masked,comparative study including 60 eyes of 30 patients(mean age 61.16±10 years)affected by POAG.Eyes were divided into 3 groups:Group 1 eyes underwent the Xen 45 Gel Stent procedure,Group 2 eyes were under medical therapy,Group 3 eyes were surgically treated by trabeculectomy.All patients underwent HRT II IVCM analysis of cornea,limbus,conjunctiva,sub-tenionian space and sclera.Results:The Xen 45 Gel stent,if properly positioned in the sub-conjunctival space preserves goblet cells and limits ocular surface inflammation.Regular corneal epithelial cells with micro-cysts,and normo-reflective sub-epithelial nerve plexus are documented by IVCM.In sub Tenon’s implants an alternative lamellar intra-scleral filtration is detectable.Combined surgical procedures show a noticeable number of inflammatory cells with rare micro-cysts.Post-trabeculectomy inflammatory reaction is more evident than Xen 45 Gel Stent associated surgical procedures,but less than medical therapy where a conspicuous presence of Langerhans cells,peri-neural infiltrates,marked loss of goblet cells and fibrosis is visible.Conclusion:Ocular surface inflammation was more notable in topical therapy than after trabeculectomy,which itself causes more inflammation than XEN Gel stents.展开更多
Background:To develop and validate a deep learning-based approach to the fully-automated analysis of macaque corneal sub-basal nerves using in vivo confocal microscopy(IVCM).Methods:IVCM was used to collect 108 images...Background:To develop and validate a deep learning-based approach to the fully-automated analysis of macaque corneal sub-basal nerves using in vivo confocal microscopy(IVCM).Methods:IVCM was used to collect 108 images from 35 macaques.58 of the images from 22 macaques were used to evaluate different deep convolutional neural network(CNN)architectures for the automatic analysis of sub-basal nerves relative to manual tracings.The remaining images were used to independently assess correlations and interobserver performance relative to three readers.Results:Correlation scores using the coefficient of determination between readers and the best CNN averaged 0.80.For inter-observer comparison,inter-correlation coefficients(ICCs)between the three expert readers and the automated approach were 0.75,0.85 and 0.92.The ICC between all four observers was 0.84,the same as the average between the CNN and individual readers.Conclusions:Deep learning-based segmentation of sub-basal nerves in IVCM images shows high to very high correlation to manual segmentations in macaque data and is indistinguishable across readers.As quantitative measurements of corneal sub-basal nerves are important biomarkers for disease screening and management,the reported work offers utility to a variety of research and clinical studies using IVCM.展开更多
The confocal microscopy technique was applied for nonlinear optical characterization of single β-barium-borate(β-BBO) nanocrystals. The experimental setup allows measurements of the laser polarization-selective se...The confocal microscopy technique was applied for nonlinear optical characterization of single β-barium-borate(β-BBO) nanocrystals. The experimental setup allows measurements of the laser polarization-selective secondharmonic(SH) generation, and the results can be used to determine the nanocrystals' c-axis orientation, as well as to obtain information about their second-order susceptibility χ^(2). The dependence of the SH signal on the laser polarization allowed the discrimination of individual particles from aggregates. The data were fitted using a model that takes into account the BBO properties and the experimental setup characteristics considering(i) the electrostatic approximation,(ii) the effects of the microscope objective used to focus the light on the sample in an epi-geometry configuration, and(iii) the symmetry of χ^(2) for the β-BBO nanocrystals. A signal at the third-harmonic frequency was also detected, but it was too weak to be studied in detail.展开更多
Spectral bleedthrough (SBT) ratio is dependent on the level of fluorescence intensity in confocal imaging. Precision Frster resonance energy transfer (FRET) algorithm corrects SBT ratio according to fluorescence i...Spectral bleedthrough (SBT) ratio is dependent on the level of fluorescence intensity in confocal imaging. Precision Frster resonance energy transfer (FRET) algorithm corrects SBT ratio according to fluorescence intensity and avoids over-or under-estimation of SBT ratio. In this letter, we propose a new method to accurately measure the FRET efficiency of FRET plasmid in single living cells by combining the calculation of SBT in precision FRET algorithm with E-FRET formulae. We also use this method to measure the FRET efficiency of FRET-Bid, and find that in healthy A549 cells it is about 15%, which is verified by FRET acceptor photobleaching method.展开更多
Thygeson superficial punctate keratitis (TSPK) is a rare,chronic,inflammatory disorder,with adverse impact on the visual function and quality of life in patients.It was first described by Thygeson in 1950,1 as typic...Thygeson superficial punctate keratitis (TSPK) is a rare,chronic,inflammatory disorder,with adverse impact on the visual function and quality of life in patients.It was first described by Thygeson in 1950,1 as typically bilateral,corneal epithelial opacities without associated stromal involvement or corneal edema.Recurrent episodes of tearing,foreign body sensation,photophobia,and reduced vision are observed.To date,virus infection and immune factors have been considered as important risk factors for visual deterioration in the patients with TSPK,but the origin of the comeal opacities remains poorly understood.Keywords:Thygeson superficial punctate keratitis ; laser confocal microscopy展开更多
Live cell imaging methods provide important insights into the dynamics of cellular processes that cannot be derived easily from population-averaged datasets.In the bioenergy field,much research is focused on fermentat...Live cell imaging methods provide important insights into the dynamics of cellular processes that cannot be derived easily from population-averaged datasets.In the bioenergy field,much research is focused on fermentation of cellulosic biomass by thermophilic microbes to produce biofuels;however,little effort is dedicated to the development of imaging tools to monitor this dynamic biological process.This is,in part,due to the experimental challenges of imaging cells under both anaerobic and thermophilic conditions.Here an imaging system is described that integrates confocal microscopy,a flow cell device,and a lipophilic dye to visualize cells.Solutions to technical obstacles regarding suitable fluorescent markers,photodamage during imaging,and maintenance of environmental conditions during imaging are presented.This system was utilized to observe cellulose colonization by Clostridium thermocellum under anaerobic conditions at 60℃.This method enables live cell imaging of bacterial growth under anaerobic and thermophilic conditions and should be widely applicable to visualizing different cell types or processes in real time.展开更多
BACKGROUND: Alpha-tocopherol ( α-tocopherol) can effectively relieve neuronal damage induced by oxygen-centered free radicals. However, the effective dose remains controversial. OBJECTIVE: To evaluate the protect...BACKGROUND: Alpha-tocopherol ( α-tocopherol) can effectively relieve neuronal damage induced by oxygen-centered free radicals. However, the effective dose remains controversial. OBJECTIVE: To evaluate the protective effects of low-concentration α-tocopherol on neuronal membranes. DESIGN, TIME AND SETTING: Contrast observation and in vitro study, performed at Laboratory of Neurosurgery, Tianjin Huanhu Hospital between April and September 2006. MATERIALS: Fetal cortical neurons were derived from two 14-day pregnant SD rats, and α-tocopherol was provided by Sigma, USA. METHODS: The neurons were randomly assigned to six groups: (1) normal: neurons were cultured under normal conditions; (2) oxidative damage: oxidative free radicals was damaged using the Fenton reaction; (3) α-tocopherol: neurons were cultured in different concentrations of -tocopherol 10, 20, 40, and 80 mg/L for 2 hours, respectively. MAIN OUTCOME MEASURES: Neuronal membrane damage was observed using a confocal laser microscope, and malonaldehyde production was detected using the thiobarbituric acid method. RESULTS: At normal, biological concentrations (10 mg/L), α-tocopherol induced no change in the damaged neurons (P 〉 0.05). However, at a concentration of 80 mg/L, the number of damaged neurons was significantly reduced, compared with the damage group (P 〈 0.05). Malonaldehyde levels following 80 mg/L α-tocopherol treatment were less than the oxygen free radical damage group (P 〈 0.05), but greater than the control group (P 〈 0.01). CONCLUSION: A concentration of 80 mg/L α-tocopherol can effectively protect the neuronal cell membrane from oxidative damage展开更多
Corneal neuromas,also termed microneuromas,refer to microscopic,irregula rly-shaped enlargements of terminal subbasal nerve endings at sites of nerve damage or injury.The formation of corneal neuromas results from dam...Corneal neuromas,also termed microneuromas,refer to microscopic,irregula rly-shaped enlargements of terminal subbasal nerve endings at sites of nerve damage or injury.The formation of corneal neuromas results from damage to corneal nerves,such as following corneal pathology or corneal or intraocular surge ries.Initially,denervated areas of sensory nerve fibers become invaded by sprouts of intact sensory nerve fibers,and later injured axons regenerate and new sprouts called neuromas develop.In recent years,analysis of corneal nerve abnormalities including corneal neuromas which can be identified using in vivo confocal microscopy,a non-invasive imaging technique with microscopic resolution,has been used to evaluate corneal neuropathy and ocular surface dysfunction.Corneal neuromas have been shown to be associated with clinical symptoms of discomfort and dryness of eyes,and are a promising surrogate biomarker for ocular surface diseases,such as neuropathic corneal pain,dry eye disease,diabetic corneal neuropathy,neurotrophic keratopathy,Sjogren's syndrome,bullous keratopathy,post-refra ctive surgery,and others.In this review,we have summarized the current literature on the association between these ocular surface diseases and the presentation of corneal microneuromas,as well as elaborated on their pathogenesis,visualization via in vivo confocal microscopy,and utility in monitoring treatment efficacy.As current quantitative analysis on neuromas mainly relies on manual annotation and quantification,which is user-dependent and labor-intensive,future direction includes the development of artificial intelligence software to identify and quantify these potential imaging biomarkers in a more automated and sensitive manner,allowing it to be applied in clinical settings more efficiently.Combining imaging and molecular biomarkers may also help elucidate the associations between corneal neuromas and ocular surface diseases.展开更多
文摘AIM: To evaluate the corneal cell morphology of new keratoconus patients wearing two different types of rigid gas-permeable(RGP) contact lenses for 1y.METHODS: Thirty nine eyes of 39 new keratoconus patients were selected and randomly fitted with two types of RGP contact lenses.Group 1 had 21 eyes with regular rigid gas-permeable(RRGP) contact lens and rest 18 eyes were in group 2 with specially designed rigid gas-permeable(SRGP) contact lens.Corneal cell morphology was evaluated using a slit scanning confocal microscope at no-lens wear and after 1y of contact lens wearing.RESULTS: After 1y of contact lens wearing in group 1,the mean anterior and posterior stromal keratocyte density were significantly less(P=0.006 and P=0.001,respectively) compared to no-lens wear.The mean cell area of anterior and posterior stromal keratocyte were also significantly different(P=0.005 and P=0.001) from no-lens wear.The anterior and posterior stromal haze increased by 18.74% and 23.81%,respectively after 1y of contact lens wearing.Whereas in group 2,statistically significant changes were observed only in cell density & area of anterior stroma(P=0.001 and P=0.001,respectively) after 1y.While,level of anterior and posterior stromal haze increased by 16.67% and 11.11% after 1y of contact lens wearing.Polymegathism and pleomorphism also increased after 1y of contact lens wearing in both the contact lens groups.CONCLUSION: Confocal microscopy observation shows the significant alterations in corneal cell morphology of keratoconic corneas wearing contact lenses especially in group 1.The type of contact lens must be carefully selected to minimize changes in corneal cell morphology.
基金Supported by the National Natural Science Foundation of China(No.81070755No.81071301)
文摘AIM: To describe the clinical features and microstructural characteristics assessed by in vivo confocal microscopy(IVCM) in patients with ocular cicatricial pemphigoid(OCP).· METHODS: A descriptive, uncontrolled case series study. Patients diagnosed with OCP were examined by clinical history, slit-lamp biomicroscopy features and IVCM images. The results of direct immunofluorescence(DIF) biopsies and indirect immunofluorescence(IIF) were also recorded. Local and systemic immunosuppressive therapy were administered and adjusted according to response.·RESULTS: A total of 12 consecutive OCP patients(7male, 5 female; mean age 60.42 ±10.39y) were recruited.All patients exhibited bilateral progressive conjunctival scarring and recurrent chronic conjunctivitis was the most frequent clinical pattern. The mean duration of symptoms prior to diagnosis of OCP was 2.95 ±2.85y(range: 5mo to 10y). The Foster classification varied from stage I to IV and 20 eyes(83%) were within or greater than Foster stage Ⅲ on presentation. Two of the 12patients(17%) demonstrated positive DIF; 3 of the 12(25%) patients reported positive IIF. The mean duration of the follow-up period was 20.17 ±11.88mo(range: 6 to48mo). IVCM showed variable degrees of abnormality in the conjuctiva-cornea and conjuctival scarring was detected in all the involved eyes. Corneal stromal cell activation and dendritic cell infiltration presented asocular surface inflammation, ocular surface keratinization along with the destroyed Vogt palisades was noted in eyes with potential limbal stem cell deficiency. After treatment, remission of ocular surface inflammation was achieved in all the patients, 18 eyes(75%) remained stable, 6 eyes(25%) had recurrent conjunctivitis and cicatrization in 2 eyes(8%) was progressing.· CONCLUSION: As an autoimmune disease, OCP manifests as variable degrees of clinical and laboratory abnormalities with both local and systemic immunosuppressive treatment playing important roles in disease therapy. IVCM can be as a valuable non-invasive technique to assess ocular surface changes in a cellular level with a potential value for providing diagnostic evidence and monitoring therapeutic effects during follow-up.
基金Research reported in this publication was supported by the National Cancer Institute Cancer Center Support Grant(P30 CA023074)and used the Tissue Acquisition and Cellular/Molecular Analysis Shared Resource at the University of Arizona.
文摘Objective:Novel optical imaging modalities are under development with the goal of obtaining an“optical biopsy”to efficiently provide pathologic details.One such modality is confocal microscopy which allows in situ visualization of cells within a layer of tissue and imaging of cellular-level structures.The goal of this study is to validate the ability of confocal microscopy to quickly and accurately differentiate between normal renal tissue and cancer.Methods:Specimens were obtained from patients who underwent robotic partial nephrectomy for renal mass.Samples of suspected normal and tumor tissue were extracted from the excised portion of the kidney and stained with acridine orange.The stained samples were imaged on a Nikon E600 C1 Confocal Microscope.The samples were then submitted for hematoxylin and eosin processing and read by an expert pathologist to provide a gold-standard diagnosis that can later be compared to the confocal images.Results:This study included 11 patients,17 tissue samples,and 118 confocal images.Of the 17 tissue samples,10 had a gold-standard diagnosis of cancer and seven were benign.Of 118 confocal images,66 had a gold-standard diagnosis of cancer and 52 were benign.Six confocal images were used as a training set to train eight observers.The observers were asked to rate the test images on a six point scale and the results were analyzed using a web based receiver operating characteristic curve calculator.The average accuracy,sensitivity,specificity,and area under the empirical receiver operating characteristic curve for this study were 91%,98%,81%,and 0.94 respectively.Conclusion:This preliminary study suggest that confocal microscopy can be used to distinguish cancer from normal tissue with high sensitivity and specificity.The observers in this study were trained quickly and on only six images.We expect even higher performance as observers become more familiar with the confocal images.
文摘Objective To study the effect of ginsenoside Rb1 and total saponin of dipsacus asper on intracellular free calcium concentration mediated by β amyloid protein.So as to lay a foundation for developing effective Chinese traditional medicine to treat Alzheimer’s disease.Methods The technique of laser scanning confocal microscopy combining primary cultured neurons was adopted to quantitatively analyze the change of [Ca 2+ ] i.Results The [Ca 2+ ] i of primary cultured hippocampal neurons was nmol·L -1 on basal levels.Control group showed obvious change of calcium vibration,[Ca 2+ ] i was elevated to nmol·L -1 .The peak of [Ca 2+ ] i of Rb1 group reached nmol·L -1 and was lower than that of control group .The tSDA group displayed distinct change of calcium vibration too,and [Ca 2+ ] i reached nmol·L -1 .There was a significant difference in [Ca 2+ ] i between control and tSDA group .Conclusion The research indicated that one of mechanisms by which Rb1 and tSDA protected the neurons was to maintain the balance of [Ca 2+ ] i.
文摘Confocal microscopy is a method which has been increasingly used over the last decade in the study of the anterior ocular surface. The method allows testing and <em>in vivo</em> high resolution imaging of the structures of the anterior eye segment, at a cellular level, which is close to the histological examination of tissues. The data provided by this method allow for a better understanding of both the functional and pathological processes occurring in the anterior ocular surface not only for scientific purposes but also in clinical practice. The aim of the present work is to summarize the current knowledge and applications of confocal microscopy of the anterior ocular surface.
基金supported by the National Natural Science Foundation of Guangdong Province(Grant No.2018A030313833)supported by the National Natural Science Foundation of Guangdong Province(Grant No.2020A1515010103)supported by the National Natural Science Foundation Incubation Program of Guangdong Provincial People’s Hospital(Grant No.KY01201147 and KY01201146).
文摘Background:To compare the efficacy of Ex-PRESS implantation versus trabeculectomy combined with phacoemulsification.Methods:A retrospective 12-month study on patients with coincident primary open-angle glaucoma(POAG)and cataract.The patients underwent combined phacoemulsification and Ex-PRESS implant(Phaco-ExPRESS,n=35)or phacotrabeculectomy(Phaco-Trab,n=35).The morphological structures of the filtering bleb were examined by slitlamp,anterior segment optical coherence tomography(AS-OCT)and in vivo confocal microscopy(IVCM).Complete success was defined as postoperative intraocular pressure(IOP)<18 mmHg without the use of anti-glaucoma medication.Qualified success was defined as postoperative IOP<18 mmHg with or without anti-glaucoma medications.The data were collected preoperatively and postoperatively at 2 weeks,1 month,3 months,6 months,and 12 months.Results:No significant difference in the variables such as age,IOP and perimetry was found between the groups of Phaco-ExPRESS and Phaco-Trab.At the one-year postoperative visit for filtering blebs,Phaco-ExPRESS increased the mean area of epithelial microcysts significantly from 0.10±0.05 to 0.20±0.09μm^(2)perμm^(2),while Phaco-Trab decreased the mean area significantly from 0.08±0.04 to 0.04±0.06μm^(2)perμm^(2).Notably,the hyperreflective dots detected by IVCM decreased by 84.9%in Phaco-ExPRESS but increased by 36.3%in Phaco-Trab.The hyperreflective dots were further identified as neutrophil-and monocyte-like cells.The number of these cells were negatively correlated with the microcysts area(r=−0.7,P<0.01)but positively associated with the grade of connective tissue(r=0.5,P<0.01).By creating different microstructural changes in the filtering blebs,Phaco-ExPRESS produced a higher complete success rate(84.9%vs.41.2%,P<0.01)and significant decrease in the number of anti-glaucoma medications(P<0.01)when compared with those in Phaco-Trab.However,the qualified success showed no significant difference between the two groups(100.0%vs.91.2%,P=0.24).Conclusions:At the one-year follow-up,Phaco-ExPRESS generated better filtering bleb with larger area of microcysts,looser connective tissues,and less inflammation than that of Phaco-Trab,providing adequate IOP control and less IOP-lowering medications.These findings indicate that Phaco-ExPRESS could be more preferred than Phaco-Trab for the treatment of patients with coincident POAG and cataract.
基金Supported by the National Natural Science Foundation of China(No.82371058)the Natural Science Foundation of Shandong Province(No.ZR2020MH172)+2 种基金the Ophthalmology New Technology Incubation Fund Program(Ophthalmology Incubation Fund Phase II Project[2022]No.[005])Medicine Science and Technology Development Program of Shandong Province(No.202107020108)Qingdao Science and Technology Beneficiary Program(No.24-1-8-smjk-16-nsh).
文摘AIM:To evaluate the effects of antiglaucoma eye drops on corneal nerves by in vivo confocal microscopy(IVCM).METHODS:This study comprised 79 patients diagnosed with glaucoma and 16 healthy control individuals.Among the glaucoma patients,54 were treated with medication,while 25 remained untreated.Central corneal images were evaluated by IVCM,and then ACCMetrics was used to calculate the following parameters:corneal nerve fiber density(CNFD),branch density(CNBD),fiber length(CNFL),total branch density(CTBD),fiber area(CNFA),fiber width(CNFW),and fractal dimension(CNFrD).The correlation between IVCM parameters and drugs was evaluated using non-parametric measurements of Spearman’s rank correlation coefficient.RESULTS:The CNFD was reduced in glaucoma groups compared to healthy subjects(P<0.01).Patients using anti-glaucoma medications exhibited poorer confocal parameters compared to untreated patients.As the number of medications and usage count increased,CNFD,CNBD,CNFL,CTBD,CNFA,and CNFrD experienced a decline,while CNFW increased(all P<0.01).For the brinzolamide-therapy group,there was a significant decrease in CNFD and CNFL compared to the other monotherapy groups(P<0.001).In the absence of medication,CNFD in males was lower than that in females(P<0.05).Among patients under medication therapy,CNFD remained consistent between males and females.CONCLUSION:Antiglaucoma eye drops affect the microstructure of corneal nerves.IVCM and ACCMetrics are useful tools that could be used to evaluate the corneal nerve changes.
基金supported by National Key R&D Program of China(grant no.2022YFC2404201)the Chinese Academy of Sciences Project for Young Scientists in Basic Research(grant no.YSBR067).
文摘Conventional microscopes designed for submicron resolution in biological research are hindered by a limited field of view,typically around 1 mm.This restriction poses a challenge when attempting to simultaneously analyze various parts of a sample,such as different brain areas.In addition,conventional objective lenses struggle to perform consistently across the required range of wavelengths for brain imaging in vivo.Here we present a novel mesoscopic objective lens with an impressive field of view of 8 mm,a numerical aperture of 0.5,and a working wavelength range from 400 to 1000 nm.We achieved a resolution of 0.74μm in fluorescent beads imaging.The versatility of this lens was further demonstrated through high-quality images of mouse brain and kidney sections in a wide-field imaging system,a confocal laser scanning system,and a two-photon imaging system.This mesoscopic objective lens holds immense promise for advancing multi-wavelength imaging of large fields of view at high resolution.
文摘Background:Dry eye patients suffer from all kinds of symptoms.Sometimes,the clinical signs evaluation does not disclose any obvious difference in routine examination;in vivo confocal microscopy (IVCM) is a powerful tool for ocular surface disease.This study aimed to clarify meibomian gland (MG) alterations in dry eye patients with different symptoms and to compare the findings using IVCM.Methods:A total of sixty patients were recruited,all subjected to Ocular Surface Disease Index (OSDI) and Salisbury Eye Evaluation Questionnaire (SEEQ),and questionnaires for the assessment of dry eye symptoms before clinical sign examinations were given to the patients.Finally,IVCM was applied to observe MG's structure.Statistical analysis was performed using the t-test,Mann-Whitney U-test and Spearman correlation analysis.The differences were statistically significant when P 〈 0.05.Results:In the severe symptom group,OSDI and SEEQ scores were significantly higher (P 〈 0.05) compared with the mild symptoms group.All other clinical sign examinations had no statistical difference in the two groups (P 〉 0.05).However,all the IVCM-observed data showed that patients with severe symptoms had more significant fibrosis in MG (acinar unit area 691.87 ± 182.01 μm^2 for the severe,992.17 ± 170.84 μm^2 for the mild;P 〈 0.05) and severer decrease in the size of MG acinar units than those observed in patients with mild symptoms (MG acinar unit density [MGAUD] 70.08 ± 18.78 glands/mm2,MG acinar unit longest diameter [MGALD] 51.50 ± 15.51 μm,MG acinar unit shortest diameter [MGASD] 20.30 ± 11.85 μm for the severe,MGAUD 89.53 ± 39.88 glands/mm2,MGALD 81.57 ± 21.14 μm,MGASD 42.37 ± 14.55 μm for the mild;P 〈 0.05).Dry eye symptoms were negatively correlated with MG confocal microscopic parameters and positively correlated with conjunctival inflammatory cells and Langerhans cells (P 〈 0.05).Conclusions:IVCM application provides a strong support to differentiate dry eye patients with different symptoms:meibomian gland dysfunction (MGD) plays a pivotal role in dry eye aggravation,and using IVCM to observe MG fibrosis,changes in size and density of MG as well as status of inflammation cells can help not only correctly diagnose the type and severity of dry eye,but also possibly prognosticate in routine eye examination in the occurrence of MGD.
文摘Objective To examine the effects of melatonin on the dynamic changes in the concentration of intracellular free Ca 2+ ([Ca 2+ ]i) in single intact cultured cortical neurons isolated from fetal rats, in order to explore the possible antiaging mechanisms of melatonin (MT) Methods Using the highly fluorescent Ca 2+ sensitive indicator Fluo 3/AM, cortical neurons cultured in a 35?mm Tissue Culture Dish were in incubated for 45?min at room temperature with 5?μmol/L Fluo 3/AM, resulting in proper intracellular dye concentration to provide adequate signal strength for detection and excellent Laser Scanning Confocal Microscopy (LSCM) imaging of [Ca 2+ ]i while not disturbing normal intracellular physiology The changes in fluorescent intensity were monitored by LSCM Results Bay K8644 (10 6 ?mol/L), KCl (20 ?mmol/L), sodium L glutamate (Glu, 50?μmol/L) caused a rapid increase of [Ca 2+ ]i in cortical neurons, and this increase could be significantly attenuated by 10 6 and 10 7 mol/L MT Conclusions MT could antagonize the extracellular Ca 2+ influx, reduce Ca 2+ overload, and have a protective effect on neurons This may be one of the important antiaging mechanisms of MT
基金This study was supported by Henan Medical Science and Technology Project(201503153)Talent project of Henan Provincial People's Hospital(23456-4).
文摘Parkinson's disease(PD)is a chronic,progressive neurodegenerative disease affecting about 2%-3% of population above the age of 65.In recent years,Parkinson's research has mainly focused on motor and non-motor symptoms while there are limited studies on neurodegeneration which is associated with balance problems and increased incidence of falls.Corneal confocal microscopy(CCM)is a real-time,non-invasive,in vivo ophthalmic imaging technique for quantifying nerve damage in peripheral neuropathies and central neurodegenerative disorders.CCM has shown significantly lower corneal nerve fiber density(CNFD)in patients with PD compared to healthy controls.Reduced CNFD is associated with decreased intraepidermal nerve fiber density in PD.This review provides an overview of the ability of CCM to detect nerve damage associated with PD.
文摘BACKGROUND Currently,there is insufficient data about the accuracy in the diagnosing of pancreatic cystic lesions(PCLs),especially with novel endoscopic techniques such as with direct intracystic micro-forceps biopsy(mFB)and needle-based confocal laser-endomicroscopy(nCLE).AIM To compare the accuracy of endoscopic ultrasound(EUS)and associated techniques for the detection of potentially malignant PCLs:EUS-guided fine needle aspiration(EUS-FNA),contrast-enhanced EUS(CE-EUS),EUS-guided fiberoptic probe cystoscopy(cystoscopy),mFB,and nCLE.METHODS This was a single-center,retrospective study.We identified patients who had undergone EUS,with or without additional diagnostic techniques,and had been diagnosed with PCLs.We determined agreement among malignancy after 24-mo follow-up findings with detection of potentially malignant PCLs via the EUSguided techniques and/or EUS-guided biopsy when available(EUS malignancy detection).RESULTS A total of 129 patients were included, with EUS performed alone in 47/129. In 82/129 patients,EUS procedures were performed with additional EUS-FNA (21/82), CE-EUS (20/82), cystoscopy(27/82), mFB (36/82), nCLE (44/82). Agreement between EUS malignancy detection and the 24-mo follow-up findings was higher when associated with additional diagnostic techniques thanEUS alone [62/82 (75.6%) vs 8/47 (17%);OR 4.35, 95%CI: 2.70-7.37;P < 0.001]. The highestmalignancy detection accuracy was reached when nCLE and direct intracystic mFB were bothperformed, with a sensitivity, specificity, positive predictive value, negative predictive value andobserved agreement of 100%, 89.4%, 77.8%, 100% and 92.3%, respectively (P < 0.001 comparedwith EUS-alone).CONCLUSIONThe combined use of EUS-guided mFB and nCLE improves detection of potentially malignantPCLs compared with EUS-alone, EUS-FNA, CE-EUS or cystoscopy.
文摘Purpose:Assessing the quality of the ocular surface by in vivo scanning laser confocal microscopy(IVCM)in primary open angle glaucoma(POAG)patients treated by Xen 45 Gel Stent,medical therapy and trabeculectomy.Methods:Retrospective,single-center,single-masked,comparative study including 60 eyes of 30 patients(mean age 61.16±10 years)affected by POAG.Eyes were divided into 3 groups:Group 1 eyes underwent the Xen 45 Gel Stent procedure,Group 2 eyes were under medical therapy,Group 3 eyes were surgically treated by trabeculectomy.All patients underwent HRT II IVCM analysis of cornea,limbus,conjunctiva,sub-tenionian space and sclera.Results:The Xen 45 Gel stent,if properly positioned in the sub-conjunctival space preserves goblet cells and limits ocular surface inflammation.Regular corneal epithelial cells with micro-cysts,and normo-reflective sub-epithelial nerve plexus are documented by IVCM.In sub Tenon’s implants an alternative lamellar intra-scleral filtration is detectable.Combined surgical procedures show a noticeable number of inflammatory cells with rare micro-cysts.Post-trabeculectomy inflammatory reaction is more evident than Xen 45 Gel Stent associated surgical procedures,but less than medical therapy where a conspicuous presence of Langerhans cells,peri-neural infiltrates,marked loss of goblet cells and fibrosis is visible.Conclusion:Ocular surface inflammation was more notable in topical therapy than after trabeculectomy,which itself causes more inflammation than XEN Gel stents.
基金This work was supported by grants R01NS097221(JDO and JLM),NS113703(JLM)and U42OD013117 from the National Institutes of Health and a Blaustein Pain Research Grant,John Hopkins University School of Medicine(JLM).
文摘Background:To develop and validate a deep learning-based approach to the fully-automated analysis of macaque corneal sub-basal nerves using in vivo confocal microscopy(IVCM).Methods:IVCM was used to collect 108 images from 35 macaques.58 of the images from 22 macaques were used to evaluate different deep convolutional neural network(CNN)architectures for the automatic analysis of sub-basal nerves relative to manual tracings.The remaining images were used to independently assess correlations and interobserver performance relative to three readers.Results:Correlation scores using the coefficient of determination between readers and the best CNN averaged 0.80.For inter-observer comparison,inter-correlation coefficients(ICCs)between the three expert readers and the automated approach were 0.75,0.85 and 0.92.The ICC between all four observers was 0.84,the same as the average between the CNN and individual readers.Conclusions:Deep learning-based segmentation of sub-basal nerves in IVCM images shows high to very high correlation to manual segmentations in macaque data and is indistinguishable across readers.As quantitative measurements of corneal sub-basal nerves are important biomarkers for disease screening and management,the reported work offers utility to a variety of research and clinical studies using IVCM.
基金support from the Instituto Nacional de Fotonica-INFoConselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq+2 种基金Coordenacao de Aperfeicoamento de Pessoal de Nível Superior-CAPESFundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco-FACEPEFundacao de Amparo a Pesquisa do Estado de Goiás-FAPEG
文摘The confocal microscopy technique was applied for nonlinear optical characterization of single β-barium-borate(β-BBO) nanocrystals. The experimental setup allows measurements of the laser polarization-selective secondharmonic(SH) generation, and the results can be used to determine the nanocrystals' c-axis orientation, as well as to obtain information about their second-order susceptibility χ^(2). The dependence of the SH signal on the laser polarization allowed the discrimination of individual particles from aggregates. The data were fitted using a model that takes into account the BBO properties and the experimental setup characteristics considering(i) the electrostatic approximation,(ii) the effects of the microscope objective used to focus the light on the sample in an epi-geometry configuration, and(iii) the symmetry of χ^(2) for the β-BBO nanocrystals. A signal at the third-harmonic frequency was also detected, but it was too weak to be studied in detail.
基金supported by the National Natural Science Foundation of China (Nos. 31071218 and 60627003)the Natural Science Foundation of Guangdong Province (Nos. 8151063101000031 and 9251063101000009)
文摘Spectral bleedthrough (SBT) ratio is dependent on the level of fluorescence intensity in confocal imaging. Precision Frster resonance energy transfer (FRET) algorithm corrects SBT ratio according to fluorescence intensity and avoids over-or under-estimation of SBT ratio. In this letter, we propose a new method to accurately measure the FRET efficiency of FRET plasmid in single living cells by combining the calculation of SBT in precision FRET algorithm with E-FRET formulae. We also use this method to measure the FRET efficiency of FRET-Bid, and find that in healthy A549 cells it is about 15%, which is verified by FRET acceptor photobleaching method.
文摘Thygeson superficial punctate keratitis (TSPK) is a rare,chronic,inflammatory disorder,with adverse impact on the visual function and quality of life in patients.It was first described by Thygeson in 1950,1 as typically bilateral,corneal epithelial opacities without associated stromal involvement or corneal edema.Recurrent episodes of tearing,foreign body sensation,photophobia,and reduced vision are observed.To date,virus infection and immune factors have been considered as important risk factors for visual deterioration in the patients with TSPK,but the origin of the comeal opacities remains poorly understood.Keywords:Thygeson superficial punctate keratitis ; laser confocal microscopy
基金supported by the BioEnergy Science Center (BESC),which is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science
文摘Live cell imaging methods provide important insights into the dynamics of cellular processes that cannot be derived easily from population-averaged datasets.In the bioenergy field,much research is focused on fermentation of cellulosic biomass by thermophilic microbes to produce biofuels;however,little effort is dedicated to the development of imaging tools to monitor this dynamic biological process.This is,in part,due to the experimental challenges of imaging cells under both anaerobic and thermophilic conditions.Here an imaging system is described that integrates confocal microscopy,a flow cell device,and a lipophilic dye to visualize cells.Solutions to technical obstacles regarding suitable fluorescent markers,photodamage during imaging,and maintenance of environmental conditions during imaging are presented.This system was utilized to observe cellulose colonization by Clostridium thermocellum under anaerobic conditions at 60℃.This method enables live cell imaging of bacterial growth under anaerobic and thermophilic conditions and should be widely applicable to visualizing different cell types or processes in real time.
基金Supported by:the 211 Key Subject Construction Foundation of Tianjin,No. 05YFGD5F02500
文摘BACKGROUND: Alpha-tocopherol ( α-tocopherol) can effectively relieve neuronal damage induced by oxygen-centered free radicals. However, the effective dose remains controversial. OBJECTIVE: To evaluate the protective effects of low-concentration α-tocopherol on neuronal membranes. DESIGN, TIME AND SETTING: Contrast observation and in vitro study, performed at Laboratory of Neurosurgery, Tianjin Huanhu Hospital between April and September 2006. MATERIALS: Fetal cortical neurons were derived from two 14-day pregnant SD rats, and α-tocopherol was provided by Sigma, USA. METHODS: The neurons were randomly assigned to six groups: (1) normal: neurons were cultured under normal conditions; (2) oxidative damage: oxidative free radicals was damaged using the Fenton reaction; (3) α-tocopherol: neurons were cultured in different concentrations of -tocopherol 10, 20, 40, and 80 mg/L for 2 hours, respectively. MAIN OUTCOME MEASURES: Neuronal membrane damage was observed using a confocal laser microscope, and malonaldehyde production was detected using the thiobarbituric acid method. RESULTS: At normal, biological concentrations (10 mg/L), α-tocopherol induced no change in the damaged neurons (P 〉 0.05). However, at a concentration of 80 mg/L, the number of damaged neurons was significantly reduced, compared with the damage group (P 〈 0.05). Malonaldehyde levels following 80 mg/L α-tocopherol treatment were less than the oxygen free radical damage group (P 〈 0.05), but greater than the control group (P 〈 0.01). CONCLUSION: A concentration of 80 mg/L α-tocopherol can effectively protect the neuronal cell membrane from oxidative damage
文摘Corneal neuromas,also termed microneuromas,refer to microscopic,irregula rly-shaped enlargements of terminal subbasal nerve endings at sites of nerve damage or injury.The formation of corneal neuromas results from damage to corneal nerves,such as following corneal pathology or corneal or intraocular surge ries.Initially,denervated areas of sensory nerve fibers become invaded by sprouts of intact sensory nerve fibers,and later injured axons regenerate and new sprouts called neuromas develop.In recent years,analysis of corneal nerve abnormalities including corneal neuromas which can be identified using in vivo confocal microscopy,a non-invasive imaging technique with microscopic resolution,has been used to evaluate corneal neuropathy and ocular surface dysfunction.Corneal neuromas have been shown to be associated with clinical symptoms of discomfort and dryness of eyes,and are a promising surrogate biomarker for ocular surface diseases,such as neuropathic corneal pain,dry eye disease,diabetic corneal neuropathy,neurotrophic keratopathy,Sjogren's syndrome,bullous keratopathy,post-refra ctive surgery,and others.In this review,we have summarized the current literature on the association between these ocular surface diseases and the presentation of corneal microneuromas,as well as elaborated on their pathogenesis,visualization via in vivo confocal microscopy,and utility in monitoring treatment efficacy.As current quantitative analysis on neuromas mainly relies on manual annotation and quantification,which is user-dependent and labor-intensive,future direction includes the development of artificial intelligence software to identify and quantify these potential imaging biomarkers in a more automated and sensitive manner,allowing it to be applied in clinical settings more efficiently.Combining imaging and molecular biomarkers may also help elucidate the associations between corneal neuromas and ocular surface diseases.