RESUMEN
The functional properties of biofilms are intimately related to their spatial architecture. Structural data are therefore of prime importance to dissect the complex social and survival strategies of biofilms and ultimately to improve their control. Confocal laser scanning microscopy (CLSM) is the most widespread microscopic tool to decipher biofilm structure, enabling noninvasive three-dimensional investigation of their dynamics down to the single-cell scale. The emergence of fully automated high content screening (HCS) systems, associated with large-scale image analysis, has radically amplified the flow of available biofilm structural data. In this contribution, we present a HCS-CLSM protocol used to analyze biofilm four-dimensional structural dynamics at high throughput. Meta-analysis of the quantitative variables extracted from HCS-CLSM will contribute to a better biological understanding of biofilm traits.
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Biopelículas , Microscopía Confocal , Biopelículas/crecimiento & desarrollo , Microscopía Confocal/métodos , Microbiología de Alimentos/métodos , Imagenología Tridimensional/métodos , Enfermedades Transmitidas por los Alimentos/microbiología , Ensayos Analíticos de Alto Rendimiento/métodos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Thiols function as antioxidants in food, prolonging shelf life and enhancing flavor. Moreover, thiols are vital biomolecules involved in enzyme activity, cellular signal transduction, and protein folding among critical biological processes. In this paper, the fluorescent probe PYL-NBD was designed and synthesized, which utilized the fluorescent molecule pyrazoline, the lysosome-targeted morpholine moiety, and the sensing moiety NBD. Probe PYL-NBD was tailored for the recognition of biothiols through single-wavelength excitation, yielding distinct fluorescence emission signals: blue for Cys, Hcy, and GSH; green for Cys, Hcy. Probe PYL-NBD exhibited rapid reaction kinetics (<10 min), distinct fluorescence response signals, and low detection limits (15.7 nM for Cys, 14.4 nM for Hcy, and 12.6 nM for GSH). Probe PYL-NBD enabled quantitative determination of Cys content in food samples and L-cysteine capsules. Furthermore, probe PYL-NBD had been successfully applied for confocal imaging with dual-channel detection of biothiols in various biological specimens, including HeLa cells, zebrafish, tumor sections, and Arabidopsis thaliana.
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Cisteína , Colorantes Fluorescentes , Análisis de los Alimentos , Glutatión , Lisosomas , Espectrometría de Fluorescencia , Pez Cebra , Humanos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Lisosomas/química , Lisosomas/metabolismo , Células HeLa , Cisteína/análisis , Animales , Análisis de los Alimentos/métodos , Glutatión/análisis , Espectrometría de Fluorescencia/métodos , Homocisteína/análisis , Arabidopsis/química , Límite de Detección , Microscopía ConfocalRESUMEN
YAG-laser interventions are associated with the risk of complications, including in the cornea. PURPOSE: This study evaluates the condition of the cornea after laser discission (LD) of secondary cataracts (SC) and laser iridectomy (LI) using corneal confocal microscopy (CCM). MATERIAL AND METHODS: Group 1 included patients with the diagnosis "Pseudophakia, secondary cataract", they underwent LD of SC. Patients of group 1 were divided into 2 subgroups depending on the initial state of the cornea: group 1A included patients with unaltered corneas; group 1B - with changes in the corneas. Group 2 included patients diagnosed with angle-closure glaucoma (ACG) or suspected ACG, they underwent LI. CCM was performed on the Heidelberg HRT-III system. Laser treatment was performed using the Nd:YAG-laser LPULSA SYL-9000, λ=1.064 µm. RESULTS: Immediately after treatment, subgroup 1A exhibited singular hyperreflective deposits and negligible endothelial cell loss (ECL). After 1 month, CMM findings revealed no changes in this subgroup. In subgroup 1B, a post-LD reduction in endothelial cell density led to increased polymegathism, decreased pleomorphism, heightened endothelial cell nucleus reflectivity, and moderate hyperreflective deposits after 1 month. In the second group, significant hyperreflective deposits of various sizes, increased nucleus reflectivity, and notable endothelial cell density reduction were observed immediately and 1 month after LI. CONCLUSION: The results of this study show that the possibility of developing corneal complications after photo destructive laser interventions is to a certain extent related to the initial state of the cornea. The risk of developing corneal damage increases with decreasing distance between the cornea and the irradiated structure. An increase in the level of laser radiation energy and its total values also contributes to damage to the cornea, which is possible with dense secondary cataracts and thick irises.
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Córnea , Láseres de Estado Sólido , Microscopía Confocal , Humanos , Femenino , Masculino , Córnea/cirugía , Córnea/patología , Córnea/diagnóstico por imagen , Córnea/efectos de la radiación , Anciano , Microscopía Confocal/métodos , Láseres de Estado Sólido/uso terapéutico , Láseres de Estado Sólido/efectos adversos , Terapia por Láser/métodos , Terapia por Láser/efectos adversos , Persona de Mediana Edad , Catarata/etiología , Catarata/diagnóstico , Glaucoma de Ángulo Cerrado/etiología , Glaucoma de Ángulo Cerrado/diagnóstico , Glaucoma de Ángulo Cerrado/cirugía , Glaucoma de Ángulo Cerrado/fisiopatología , Iridectomía/métodos , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/prevención & control , Extracción de Catarata/efectos adversos , Extracción de Catarata/métodos , Segmento Anterior del Ojo/diagnóstico por imagenRESUMEN
Line-field confocal optical coherence tomography (LC-OCT) is a new technology for skin cancer diagnostics. However, the interobserver agreement (IOA) of known image markers of keratinocyte carcinomas (KC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), as well as precursors, SCC in situ (CIS) and actinic keratosis (AK), remains unexplored. This study determined IOA on the presence or absence of 10 key LC-OCT image markers of KC and precursors, among evaluators new to LC-OCT with different levels of dermatologic imaging experience. Secondly, the frequency and association between reported image markers and lesion types, was determined. Six evaluators blinded to histopathologic diagnoses, assessed 75 LC-OCT images of KC (21 SCC; 21 BCC), CIS (12), and AK (21). For each image, evaluators independently reported the presence or absence of 10 predefined key image markers of KCs and precursors described in an LC-OCT literature review. Evaluators were stratified by experience-level as experienced (3) or novices (3) based on previous OCT and reflectance confocal microscopy usage. IOA was tested for all groups, using Conger's kappa coefficient (κ). The frequency of reported image marker and their association with lesion-types, were calculated as proportions and odds ratios (OR), respectively. Overall IOA was highest for the image markers lobules (κ = 0.68, 95% confidence interval (CI) 0.57;0.78) and clefting (κ = 0.63, CI 0.52;0.74), typically seen in BCC (94%;OR 143.2 and 158.7, respectively, p < 0.001), followed by severe dysplasia (κ = 0.42, CI 0.31;0.53), observed primarily in CIS (79%;OR 7.1, p < 0.001). The remaining seven image-markers had lower IOA (κ = 0.06-0.32) and were more evenly observed across lesion types. The lowest IOA was noted for a well-defined (κ = 0.07, CI 0;0.15) and interrupted dermal-epidermal junction (DEJ) (κ = 0.06, CI -0.002;0.13). IOA was higher for all image markers among experienced evaluators versus novices. This study shows varying IOA for 10 key image markers of KC and precursors in LC-OCT images among evaluators new to the technology. IOA was highest for the assessments of lobules, clefting, and severe dysplasia while lowest for the assessment of the DEJ integrity.
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Carcinoma Basocelular , Carcinoma de Células Escamosas , Queratinocitos , Queratosis Actínica , Variaciones Dependientes del Observador , Neoplasias Cutáneas , Tomografía de Coherencia Óptica , Humanos , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/diagnóstico , Tomografía de Coherencia Óptica/métodos , Carcinoma Basocelular/diagnóstico por imagen , Carcinoma Basocelular/patología , Carcinoma Basocelular/diagnóstico , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/patología , Queratinocitos/patología , Queratosis Actínica/diagnóstico por imagen , Queratosis Actínica/patología , Queratosis Actínica/diagnóstico , Microscopía Confocal/métodos , Lesiones Precancerosas/diagnóstico por imagen , Lesiones Precancerosas/patología , Femenino , Masculino , Anciano , Persona de Mediana EdadRESUMEN
The primary aim of this study was to investigate the impact of treatment with low-temperature plasma (LTP) for varying exposure durations on a multispecies cariogenic biofilm comprising C. albicans, L. casei, and S. mutans, as well as on single-species biofilms of L. casei and C. albicans, cultured on hydroxyapatite discs. Biofilms were treated with LTP-argon at a 10 mm distance for 30 s, 60 s, and 120 s. Chlorhexidine solution (0.12%) and NaCl (0.89%) were used as positive (PC) and negative controls (NC), respectively. Argon flow only was also used as gas flow control (F). Colony-forming units (CFU) recovery and confocal laser scanning microscopy (CLSM) were used to analyze biofilm viability. LTP starting at 30 s of application significantly reduced the viability of multispecies biofilms by more than 2 log10 in all treated samples (p < 0.0001). For single-species biofilms, L. casei showed a significant reduction compared to PC and NC of over 1 log10 at all exposure times (p < 0.0001). In the case of C. albicans biofilms, LTP treatment compared to PC and NC resulted in a significant decrease in bacterial counts when applied for 60 and 120 s (1.55 and 1.90 log10 CFU/mL, respectively) (p < 0.0001). A significant effect (p ≤ 0.05) of LTP in single-species biofilms was observed to start at 60 s of LTP application compared to F, suggesting a time-dependent effect of LTP for the single-species biofilms of C. albicans and L. casei. LTP is a potential mechanism in treating dental caries by being an effective anti-biofilm therapy of both single and multispecies cariogenic biofilms.
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Biopelículas , Candida albicans , Gases em Plasma , Streptococcus mutans , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Gases em Plasma/farmacología , Candida albicans/fisiología , Candida albicans/efectos de los fármacos , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/fisiología , Caries Dental/microbiología , Caries Dental/terapia , Lacticaseibacillus casei/fisiología , Humanos , Viabilidad Microbiana/efectos de los fármacos , Microscopía Confocal , FríoRESUMEN
Embryo quality assessment by optical imaging is increasing in popularity. Among available optical techniques, light sheet microscopy has emerged as a superior alternative to confocal microscopy due to its geometry, enabling faster image acquisition with reduced photodamage to the sample. However, previous assessments of photodamage induced by imaging may have failed to measure more subtle impacts. In this study, we employed DNA damage as a sensitive indicator of photodamage. We use light sheet microscopy with excitation at a wavelength of 405 nm for imaging embryo autofluorescence and compare its performance to laser scanning confocal microscopy. At an equivalent signal-to-noise ratio for images acquired with both modalities, light sheet microscopy reduced image acquisition time by ten-fold, and did not induce DNA damage when compared to non-imaged embryos. In contrast, imaging with confocal microscopy led to significantly higher levels of DNA damage within embryos and had a higher photobleaching rate. Light sheet imaging is also capable of inducing DNA damage within the embryo but requires multiple cycles of volumetric imaging. Collectively, this study confirms that light sheet microscopy is faster and safer than confocal microscopy for imaging live embryos, indicating its potential as a label-free diagnostic for embryo quality.
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Daño del ADN , Embrión de Mamíferos , Microscopía Confocal , Microscopía Confocal/métodos , Animales , Ratones , Femenino , Imagen Óptica/métodosRESUMEN
OBJECTIVE: Microsporidial stromal keratitis (MSK) is an uncommon disease. Only several case series have been reported. We aimed to describe the clinical manifestations, histopathology and treatment outcomes of MSK. METHODS AND ANALYSIS: Retrospective data of MSK diagnosed between January 2009 and December 2020 at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand were retrieved. The diagnosis was made based on corneal scraping, corneal biopsy and corneal button histopathology findings. Detailed clinical characteristics, histopathological findings and treatment outcomes were reviewed and analysed. RESULTS: 21 patients with MSK with a mean age of 63.8 years (SD 12.2) had an indolent disease onset with a median of 9 months (IQR 2.2-12.0). Five patients (23.8%) experienced ocular traumas. Herpes stromal keratitis was the most common preliminary diagnosis (33.3%), followed by non-specific ulcers and fungal keratitis. The most common corneal finding was multifocal grey-white lesions with anterior to mid-stromal infiltration and fluffy borders (66.7%). Pathogens were identified by modified trichrome staining of corneal scrapings in 11 of 14 cases (78.6%). Histopathological examination showed positive Ziehl-Neelsen staining in 17 of 19 cases (89.5%). All patients received surgical treatment, with 18 receiving therapeutic penetrating keratoplasty (TPK), 2 undergoing deep anterior lamellar keratoplasty and 1 undergoing femtosecond laser-assisted anterior lamellar keratoplasty. The overall cure rate was 76.2% after the first surgery and 95.2% after the second surgery. CONCLUSION: MSK can be easily underdiagnosed. Clues to diagnosis included a history of chronic refractory stromal infiltration and typical corneal findings of deep stromal infiltration, without epithelial defects. TPK is the preferred treatment for MSK.
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Sustancia Propia , Infecciones Fúngicas del Ojo , Queratitis , Microscopía Confocal , Microsporidiosis , Humanos , Estudios Retrospectivos , Masculino , Persona de Mediana Edad , Femenino , Infecciones Fúngicas del Ojo/patología , Infecciones Fúngicas del Ojo/microbiología , Infecciones Fúngicas del Ojo/terapia , Infecciones Fúngicas del Ojo/diagnóstico , Microsporidiosis/patología , Microsporidiosis/cirugía , Sustancia Propia/patología , Sustancia Propia/microbiología , Sustancia Propia/cirugía , Anciano , Queratitis/microbiología , Queratitis/patología , Queratitis/diagnóstico , Queratitis/terapia , Antifúngicos/uso terapéutico , Resultado del Tratamiento , Adulto , Trasplante de Córnea , Tailandia/epidemiología , BiopsiaRESUMEN
Diabetic peripheral neuropathy (DPN) is one of the most common chronic complications of diabetes. As a new detection method for DPN, corneal confocal microscopy (CCM) is characterised by rapid, non-invasive, sensitive, and quantitative characteristics, as well as good repeatability. By detecting changes in the corneal nerves, DPN can be diagnosed early, and the severity of neuropathy evaluated. It is currently an ideal DPN evaluation method and has good clinical application prospects. This paper reviews the application and progress of CCM in the evaluation of DPN and summarises the evaluation methods of CCM, corneal nerve, and DPN to provide new ideas for the clinical diagnosis and treatment of DPN.
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Córnea , Neuropatías Diabéticas , Microscopía Confocal , Humanos , Microscopía Confocal/métodos , Neuropatías Diabéticas/diagnóstico , Neuropatías Diabéticas/diagnóstico por imagen , Córnea/patología , Córnea/diagnóstico por imagen , Córnea/inervaciónRESUMEN
BACKGROUND: Epidemiological and toxicological studies indicate that increased exposure to air pollutants can lead to neurodegenerative diseases. To further confirm this relationship, we evaluated the association between exposure to ambient air pollutants and corneal nerve measures as a surrogate for neurodegeneration, using corneal confocal microscopy. METHODS: We used population-based observational cross-sectional data from The Maastricht Study including N = 3635 participants (mean age 59.3 years, 51.6% were women, and 19.9% had type 2 diabetes) living in the Maastricht area. Using the Geoscience and hEalth Cohort COnsortium (GECCO) data we linked the yearly average exposure levels of ambient air pollutants at home address-level [particulate matter with diameters of ≤ 2.5 µm (PM2.5), and ≤ 10.0 µm (PM10), nitrogen dioxide (NO2), and elemental carbon (EC)]. We used linear regression analysis to study the associations between Z-score for ambient air pollutants concentrations (PM2.5, PM10, NO2, and EC) and Z-score for individual corneal nerve measures (corneal nerve bifurcation density, corneal nerve density, corneal nerve length, and fractal dimension). RESULTS: After adjustment for potential confounders (age, sex, level of education, glucose metabolism status, corneal confocal microscopy lag time, inclusion year of participants, smoking status, and alcohol consumption), higher Z-scores for PM2.5 and PM10 were associated with lower Z-scores for corneal nerve bifurcation density, nerve density, nerve length, and nerve fractal dimension [stß (95% CI): PM2.5 -0.10 (-0.14; -0.05), -0.04 (-0.09; 0.01), -0.11 (-0.16; -0.06), -0.20 (-0.24; -0.15); and PM10 -0.08 (-0.13; -0.03), -0.04 (-0.09; 0.01), -0.08 (-0.13; -0.04), -0.17 (-0.21; -0.12)], respectively. No associations were found between NO2 and EC and corneal nerve measures. CONCLUSIONS: Our population-based study demonstrated that exposure to higher levels of PM2.5 and PM10 are associated with higher levels of corneal neurodegeneration, estimated from lower corneal nerve measures. Our results suggest that air pollution may be a determinant for neurodegeneration assessed in the cornea and may impact the ocular surface health as well.
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Contaminantes Atmosféricos , Córnea , Exposición a Riesgos Ambientales , Material Particulado , Humanos , Femenino , Material Particulado/análisis , Material Particulado/efectos adversos , Masculino , Estudios Transversales , Persona de Mediana Edad , Córnea/inervación , Contaminantes Atmosféricos/análisis , Contaminantes Atmosféricos/efectos adversos , Exposición a Riesgos Ambientales/efectos adversos , Anciano , Países Bajos/epidemiología , Adulto , Microscopía ConfocalRESUMEN
BACKGROUND: More efficient methods to detect and treat precancerous lesions of the cervix at a single visit, such as low-cost confocal microscopy, could improve early diagnosis and hence outcomes. We piloted a prototype smartphone-compatible confocal micro-endoscope (SCME) among women presenting to a public cervical cancer screening clinic in Kampala, Uganda. We describe the piloting of the SCME device at an urban clinic used by lower cadre staff. METHODS: We screened women aged 18 and 60 years, who presented for cervical cancer screening at the Kawempe National Referral Hospital Kampala, and evaluated the experience of their providers (nurses). Nurses received a 2-day training by the study doctors on how to use the SCME, which was added to the standard Visual Inspection with Acetic acid (VIA)-based cervical cancer screening. The SCME was used to take colposcopy images before and after VIA at positions 12 and 6 O'clock if VIA negative, and on precancer-suspicious lesions if VIA positive. We used questionnaires to assess the women's experiences after screening, and the experience of the nurses who operated the SCME. RESULTS: Between November 2021 and July 2022, we screened 291 women with a median age of 36 years and 65.7% were HIV positive. Of the women screened, 146 were eligible for VIA, 123 were screened with the SCME, and we obtained confocal images from 103 women. Of those screened with the SCME, 60% found it comfortable and 81% were willing to screen again with it. Confocal images from 79% of the women showed distinguishable cellular features, while images from the remaining 21% were challenging to analyze. Nurses reported a mean score of 85% regarding the SCME's usefulness to their work, 71% regarding their satisfaction and willingness to use it again, 63% in terms of ease of use, and 57% concerning the ease of learning how to operate the SCME. CONCLUSION: Our findings demonstrate the feasibility of using the SCME by lower cadre staff in low-resource settings to aid diagnosis of precancerous lesions. However, more work is needed to make it easier for providers to learn how to operate the SCME and capture high-quality confocal images.
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Colposcopía , Detección Precoz del Cáncer , Microscopía Confocal , Teléfono Inteligente , Neoplasias del Cuello Uterino , Humanos , Femenino , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Detección Precoz del Cáncer/métodos , Uganda , Persona de Mediana Edad , Microscopía Confocal/métodos , Colposcopía/métodos , Adulto Joven , Adolescente , Proyectos Piloto , Configuración de Recursos LimitadosRESUMEN
OBJECTIVES: Dental caries result from a microbial imbalance in the oral cavity. Probiotics ecologically modulate the oral microflora to prevent caries. This study evaluated the anti-cariogenic effects of two Lacticaseibacillus rhamnosus strains in vitro and in vivo to provide a more theoretical basis for its clinical applications in caries prevention. METHODS: In the study, cariogenic biofilms were grown with L. rhamnosus (LGG) or L. rhamnosus ATCC 7469 and analyzed. Quantitative real-time PCR (qPCR), Scanning Electron Microscope (SEM), and Confocal laser scanning microscope (CLSM) were used to detect the changes in the composition and architectures; cariogenic activity was measured by the lactic acid production and Transverse Microradiography (TMR). The effects of LGG on the 12 Sprague-Dawley rat caries model were assessed using Keyes scores and micro-CT analysis. Oral microbiome changes were evaluated through 16S rRNA gene high-throughput sequencing. RESULTS: L. rhamnosus can reduce cariogenic bacteria in biofilm by 14.7 % to 48.9 %, with LGG exhibiting more potent inhibitory effects. Both strains of L. rhamnosus can adhere to the surface of biofilms, reduce the extracellular polysaccharides (EPS) matrix, and loosen the biofilm structure. L. rhamnosus inhibited cariogenic activity by reducing the lactic acid production in biofilms. The bovine enamel blocks presented lower mineral loss values and lesion depth values in the group Core+L.rh and Core+LGG. LGG-ingested rats had significantly lower levels of moderate dentin lesions and higher mineral density than the control group. The 16 s rRNA gene sequencing revealed that LGG regulated the beta diversity of the oral microbial community in the rat dental caries model. CONCLUSIONS: This study revealed the promising potential of L. rhamnosus, especially the LGG strain, in the ecological prevention of dental caries. CLINICAL SIGNIFICANCE: Probiotics may provide a strategy for preventing caries by regulating the oral microecological balance. The study revealed the promising anti-caries potential of the LGG probiotic strain in vivo and in vitro. It is expected that LGG could be used as an oral probiotic for the clinical prevention and treatment of caries.
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Biopelículas , Caries Dental , Modelos Animales de Enfermedad , Lacticaseibacillus rhamnosus , Probióticos , Ratas Sprague-Dawley , Animales , Caries Dental/microbiología , Caries Dental/prevención & control , Biopelículas/efectos de los fármacos , Probióticos/uso terapéutico , Ratas , Ácido Láctico/metabolismo , ARN Ribosómico 16S , Microscopía Electrónica de Rastreo , Microtomografía por Rayos X , Esmalte Dental , Reacción en Cadena en Tiempo Real de la Polimerasa , Microbiota , Boca/microbiología , Microscopía Confocal , Microrradiografía , MasculinoRESUMEN
OBJECTIVE: To investigate caries preventive effects of 38 % silver diamine fluoride (SDF) pretreatment on neighboring tooth proximal to glass ionomer cement (GIC), including conventional GIC (CGIC) and resin-modified GIC (RMGIC) restorations in an in vitro model. METHODS: HUMAN TOOTH BLOCKS WERE RESTORED WITH: SDF+CGIC (Group 1), CGIC (Group 2), SDF+RMGIC (Group 3) or RMGIC (Group 4). Enamel specimen simulating proximal surface of neighboring tooth was placed in proximity to the restorations. The specimen underwent cariogenic challenge with cross-kingdom biofilm of Streptococcus mutans, Lacticaseibacillus casei and Candida albicans. After cariogenic challenge, the biofilm's growth kinetics, viability, and morphology were evaluated by propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR), confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. The enamel lesion depth, surface morphology and crystal characteristics were determined by micro-computed tomography (micro-CT), SEM and X-ray diffraction (XRD), respectively. RESULTS: PMA-qPCR demonstrated lower microbial growth in Group 1 and 3 compared with Group 2 and 4 (p < 0.05). CLSM showed the dead-to-live ratio in Groups 1-4 were 1.15±0.12, 0.53±0.13, 1.10±0.24 and 0.63±0.10, respectively (Group 1,3 > 2,4, p < 0.05). SEM revealed Groups 1 and 3 had scattered biofilm whereas Group 2 and 4 had confluent biofilm. Micro-CT showed the enamel lesion depths (µm) were 98±9, 126±7, 103±6 and 128±7 for Group 1 to 4, respectively (Group 1,3 < 2,4, p < 0.05). SEM revealed oriented and ordered enamel prismatic patterns in Group 1 and 3, not in Group 2 and 4. XRD showed the reflections of hydroxyapatite in Groups 1 and 3 were sharper than Groups 2 and 4. CONCLUSION: SDF pretreatment enhances the preventive effect of GIC on proximal enamel surface on neighboring tooth through inhibiting cariogenic biofilm, reducing enamel demineralization and promoting enamel remineralization. CLINICAL SIGNIFICANCE: SDF pretreatment of GIC restorations can help prevent caries on neighboring teeth, particular for patients with high caries risk.
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Biopelículas , Cariostáticos , Caries Dental , Esmalte Dental , Fluoruros Tópicos , Cementos de Ionómero Vítreo , Microscopía Electrónica de Rastreo , Compuestos de Amonio Cuaternario , Compuestos de Plata , Streptococcus mutans , Compuestos de Plata/uso terapéutico , Compuestos de Plata/farmacología , Humanos , Cementos de Ionómero Vítreo/uso terapéutico , Cementos de Ionómero Vítreo/farmacología , Caries Dental/prevención & control , Caries Dental/microbiología , Compuestos de Amonio Cuaternario/farmacología , Compuestos de Amonio Cuaternario/uso terapéutico , Biopelículas/efectos de los fármacos , Fluoruros Tópicos/uso terapéutico , Fluoruros Tópicos/farmacología , Cariostáticos/uso terapéutico , Cariostáticos/farmacología , Esmalte Dental/efectos de los fármacos , Streptococcus mutans/efectos de los fármacos , Restauración Dental Permanente/métodos , Microtomografía por Rayos X , Candida albicans/efectos de los fármacos , Difracción de Rayos X , Microscopía ConfocalRESUMEN
OBJECTIVE: To investigate the anticaries effects of graphene oxide (GO) and graphene quantum dots (GQDs) combined with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on enamel in a biofilm-challenged environment. MATERIAL AND METHODS: GO and GQDs were synthesised using citric acid. The antibiofilm and biofilm inhibition effects for Streptococcus mutans were evaluated by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and colony-forming units (CFU). Remineralisation ability was determined by assessing mineral loss, calcium-to-phosphorus ratio, and surface morphology. To create a biofilm-challenged environment, enamel blocks were immersed in S. mutans to create the lesion and then subjected to artificial saliva/biofilm cycling for 7 days. Anticaries effects of GO, GQDs, GQDs@CPP-ACP, GO@CPP-ACP, and CPP-ACP were determined by broth pH and mineral changes after 7-day pH cycling. Biocompatibility was tested using a Cell Counting Kit-8 (CCK8) assay for human gingival fibroblasts (HGF-1). RESULTS: GQDs and GO presented significant antibiofilm and biofilm inhibition effects compared to the CPP-ACP and control groups (P < 0.05). The enamel covered by GQDs and GO showed better crystal structure formation and less mineral loss (P < 0.05) than that covered by CPP-ACP alone. After 7 days in the biofilm-challenged environment, the GO@CPP-ACP group showed less lesion depth than the CPP-ACP and control groups (P < 0.05). GO and GQDs showed good biocompatibility compared to the control group by CCK8 (P > 0.05) within 3 days. CONCLUSION: GO and GQDs could improve the anti-caries effects of CPP-ACP, and CPP-ACP agents with GO or GQDs could be a potential option for enamel lesion management. CLINICAL SIGNIFICANCE: GO and GQDs have demonstrated the potential to significantly enhance the anticaries effects of CPP-ACP. Incorporating these nanomaterials into CPP-ACP formulations could provide innovative and effective options for the management of enamel lesions, offering improved preventive and therapeutic strategies in dental care.
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Biopelículas , Caseínas , Caries Dental , Esmalte Dental , Grafito , Puntos Cuánticos , Streptococcus mutans , Grafito/química , Grafito/farmacología , Biopelículas/efectos de los fármacos , Caseínas/farmacología , Esmalte Dental/efectos de los fármacos , Streptococcus mutans/efectos de los fármacos , Humanos , Caries Dental/microbiología , Caries Dental/prevención & control , Microscopía Electrónica de Rastreo , Remineralización Dental/métodos , Cariostáticos/farmacología , Cariostáticos/química , Microscopía Confocal , Concentración de Iones de Hidrógeno , Ácido Cítrico/farmacología , Fibroblastos/efectos de los fármacos , Saliva Artificial/químicaRESUMEN
Purpose: The purpose of this study was to determine the association between corneal images provided by in vivo confocal microscopy (IVCM) with clinical parameters and conjunctival expression of HLA-DR antigen in patients with dry eye disease (DED). Methods: Two hundred fourteen eyes of 214 patients with DED were analyzed, consisting of 2 groups of patients - 63 with autoimmune dry eye disease (AIDED) and 151 with non-autoimmune dry eye disease (NAIDED). Patients underwent a full clinical examination, including symptom screening, using the Ocular Surface Disease Index (OSDI) questionnaire, and objective analysis of DED signs by Schirmer's testing, tear break-up time (TBUT), Oxford's test, and IVCM corneal imaging. The IVCM scoring criteria were based on corneal sub-basal nerve density (ND), nerve morphology (NM), and inflammatory cell (IC) density. Quantification of conjunctival HLA-DR antigen was performed by flow cytometry. Results: The total IVCM score (T-IVCM) as well as the IVCM-IC subscore (sc) were positively correlated with HLA-DR levels with r = 0.3, P < 0.001 and r = 0.3, P < 0.01, respectively in the total population of patients with DED. The IVCM-NDsc was negatively correlated with TBUT in patients with AIDED (r = -0.2, P < 0.05) and with the Schirmer's test in patients with NAIDED (r = -0.24, P < 0.05). However, the IVCM-NMsc was positively correlated with the Oxford score only in patients with AIDED (r = 0.3, P < 0.05). Conclusions: The proposed IVCM scoring system showed significant correlations with clinical parameters along with conjunctival HLA-DR quantification in patients with DED. Translational Relevance: The IVCM grading score represents an interesting point of commonality among clinical parameters, imaging, and molecular investigation of the ocular surface.
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Conjuntiva , Córnea , Síndromes de Ojo Seco , Antígenos HLA-DR , Microscopía Confocal , Humanos , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/patología , Síndromes de Ojo Seco/diagnóstico , Masculino , Femenino , Antígenos HLA-DR/metabolismo , Persona de Mediana Edad , Conjuntiva/patología , Conjuntiva/metabolismo , Córnea/patología , Córnea/inervación , Córnea/metabolismo , Córnea/diagnóstico por imagen , Anciano , Adulto , Imagen Multimodal/métodos , Citometría de Flujo/métodos , Lágrimas/metabolismoRESUMEN
PURPOSE: To assess the long-term (1-year) effect of myopic femtosecond laser-assisted in situ keratomileusis (FSLASIK) on clinical characteristics and tear film biomarkers. METHODS: Eighty eyes from 80 patients who underwent FSLASIK were evaluated. Ocular surface symptoms and signs were evaluated using specific questionnaires and tests. The corneal nerves and dendritic cells were examined using in vivo confocal microscopy. Corneal sensitivity was evaluated using a Cochet-Bonnet esthesiometer. Tear inflammatory cytokines and neuropeptides were evaluated using Luminex immunoassay. These examinations were performed preoperatively and at 1, 3, 6, and 12 months postoperatively. RESULTS: Seventy-three participants completed all follow-up visits. Following FS-LASIK, ocular symptoms and signs (except Schirmer I test) worsened at 1 month but corneal and conjunctival stainings improved by 3 months. The numbers of dendritic cells and activated dendritic cells increased at the 3-month postoperative visit and recovered to preoperative levels by the 6-month visit. Ocular symptoms and corneal sensitivity recovered to preoperative levels at the 12-month visit. Tear break-up time and corneal nerve morphology were not recovered to preoperative status at the 12-month visit. Interleukin (IL)-1ß, IL-17A, tumor necrosis factor-α, and substance P tear levels significantly increased at all postoperative visits compared to preoperative levels. Corneal staining scores positively correlated with tear IL-1ß and IL-17A levels, whereas corneal nerve morphology positively correlated with corneal sensitivity and negatively correlated with substance P levels. CONCLUSIONS: Although most clinical variables improved at 12 months postoperatively, some tear inflammatory cytokines and substance P remain altered beyond 12 months, indicating that ocular homeostasis is not completely recovered. [J Refract Surg. 2024;40(8):e508-e519.].
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Biomarcadores , Córnea , Queratomileusis por Láser In Situ , Láseres de Excímeros , Miopía , Lágrimas , Humanos , Lágrimas/metabolismo , Queratomileusis por Láser In Situ/métodos , Estudios Prospectivos , Masculino , Adulto , Femenino , Miopía/cirugía , Miopía/fisiopatología , Miopía/metabolismo , Estudios de Seguimiento , Biomarcadores/metabolismo , Córnea/inervación , Córnea/metabolismo , Láseres de Excímeros/uso terapéutico , Microscopía Confocal , Adulto Joven , Citocinas/metabolismo , Agudeza Visual/fisiología , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/fisiopatología , Síndromes de Ojo Seco/diagnóstico , Síndromes de Ojo Seco/etiología , Encuestas y Cuestionarios , Persona de Mediana Edad , Proteínas del Ojo/metabolismo , Células Dendríticas/metabolismoRESUMEN
The aim of this study is to evaluate the effect of different irrigation activation methods on root canal sealer penetration in teeth with simulated internal root resorption (IRR) and calcium hydroxide (CH) applied using a confocal laser scanning microscope (CLSM). 60 incisors with a single root and a single canal were included in the study. IRR cavities were created in the middle third of the root canal, and CH was placed. The samples were randomly divided into 4 groups (n = 15) according to the irrigation activation method to be tested: standard needle irrigation (SNI), sonic activation (EDDY), photon-induced photoacoustic flow (PIPS), and shock wave enhanced emission photoacoustic flow (SWEEPS). After irrigation activation applications, the root canals were obturated. Sections of 1.0 ± 0.1 mm were taken from the apical, middle, and coronal regions of each sample. The penetration area (µm2) and maximum penetration depth (µm) of the root canal sealer were examined by CLSM and analyzed using ImageJ software. Statistical analysis was performed with a one-way ANOVA and post-hoc Tukey test at the P < 0.05 significance level. Among all irrigation activation methods tested, both the penetration area and maximum penetration depth of the root canal sealer were greater in the coronal region than in the apical region (p < 0.05). In the IRR region, there was no difference in terms of maximum penetration depth between PIPS and SWEEPS (p > 0.05), it was highest in SWEEPS (p < 0.05). PIPS and SWEEPS were better than other irrigation activation methods in the penetration of root canal sealer in the resorption areas of teeth with IRR.
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Hidróxido de Calcio , Microscopía Confocal , Resorción Radicular , Humanos , Materiales de Obturación del Conducto Radicular , Irrigación Terapéutica/métodos , Irrigación Terapéutica/instrumentación , Irrigantes del Conducto Radicular/administración & dosificación , Preparación del Conducto Radicular/métodos , Preparación del Conducto Radicular/instrumentación , IncisivoRESUMEN
OBJECTIVE: Ex vivo skin has been used to study various skin conditions from atopic dermatitis to burn injury. The aim of this research is to identify a more effective barrier improvement strategy and to evaluate topical formulations in replenishing the skin. The skin can create new longer chain fatty acids and ceramides (CERs) from topically applied skin natural fatty acid to help renew the skin's barrier. METHODS: An ex vivo skin model damaged by sequential tape stripping of the stratum corneum (SC) was used to investigate the repair of the SC. Confocal laser scanning microscopy was used to assess the SC layers recovered. Ultrastructural analysis was performed using transmission electron microscopy to visualize the lamellar bodies and intercellular lipid lamellae. RESULTS: The data in this study provide the first direct ex vivo evidence comparing different marketed formulations containing three CERs with those containing fatty acids. Free fatty acid (FFA)-containing formulations, but not CER-containing formulations, directly applied to the damaged skin, showed an increased number of repaired SC layers and this was reflected at the ultrastructural level by an increased intercellular lipid lamellae length and an increased number of lamellar bodies. CONCLUSION: These findings demonstrate that FFA-containing formulations can repair damaged ex vivo skin and point to a repair mechanism in which topically applied palmitic and stearic acids, (which boost lipid levels and elongation) can increase the production and transport of lipids into a repaired SC and thus rebuild an effective skin barrier.
OBJECTIF: La peau ex vivo a été utilisée pour étudier diverses affections cutanées, allant de la dermatite atopique aux brûlures. L'objectif de cette étude est d'identifier une stratégie d'amélioration de la barrière cutanée plus efficace et d'évaluer les formulations topiques pour reconstituer la peau. La peau peut créer de nouveaux acides gras à chaîne plus longue et des céramides (CER) à partir d'acides gras naturels de la peau appliqués par voie topique pour aider à renouveler la barrière cutanée. MÉTHODES: Un modèle de peau ex vivo endommagé par un décapage séquentiel de la couche cornée a été utilisé pour étudier la réparation de la couche cornée. La microscopie confocale à balayage laser a été utilisée pour évaluer les couches de la couche cornée récupérées. Une analyse ultrastructurale a été réalisée par microscopie électronique à transmission pour visualiser les corps lamellaires et les lamelles lipidiques intercellulaires. RÉSULTATS: Les données de cette étude fournissent les premières preuves directes ex vivo comparant différentes formulations commercialisées contenant trois CER avec celles contenant des acides gras. Les formulations contenant des acides gras libres (AGL), mais pas celles contenant des CER, appliquées directement sur la peau endommagée, ont montré un nombre accru de couches de la couche cornée réparées, ce qui s'est traduit au niveau ultrastructural par une augmentation de la longueur des lamelles lipidiques intercellulaires et une augmentation du nombre de corps lamellaires. CONCLUSION: Ces résultats démontrent que les formulations contenant des AGL peuvent réparer la peau ex vivo endommagée et indiquent un mécanisme de réparation dans lequel les acides palmitique et stéarique appliqués par voie topique (qui stimulent les taux de lipides et leur allongement) peuvent augmenter la production et le transport de lipides dans une couche cornée réparée et ainsi reconstruire une barrière cutanée efficace.
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Ácidos Grasos , Piel , Piel/metabolismo , Piel/efectos de los fármacos , Administración Tópica , Humanos , Microscopía Confocal , Microscopía Electrónica de TransmisiónRESUMEN
Mitochondria-targeted Keima (mt-Keima) is a pH-sensitive, acid-stable fluorescent protein used for the quantification of mitophagy. Mt-Keima contains a mitochondrial matrix targeting sequence and has bimodal excitation with peaks at 440 nM in neutral environments and 586 nM in acidic environments. From this bimodal excitation, a ratiometric signal may be calculated to quantify mitophagy in live cells. This chapter describes procedures for measuring mitophagy by flow cytometry and live cell confocal microscopy with mt-Keima.
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Citometría de Flujo , Microscopía Confocal , Mitocondrias , Mitofagia , Humanos , Mitocondrias/metabolismo , Microscopía Confocal/métodos , Citometría de Flujo/métodos , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/genética , Células HeLa , Concentración de Iones de HidrógenoRESUMEN
Agrobacterium-mediated transient expression is a flexible and efficient technique for introducing genes into plants, allowing for rapid and temporary gene expression. Agroinfiltration of Arabidopsis seedlings is a newly developed Agrobacterium-based transient expression system. The expression of target genes and the localization of relevant proteins can be observed within 3 days using this method. In this chapter, we present the detailed protocol for transient transformation in Arabidopsis thaliana seedlings utilizing vacuum infiltration of Agrobacterium. This procedure enables rapid and temporary gene expression by introducing exogenous DNA into Arabidopsis seedlings, particularly in easily accessible tissues such as cotyledons. This protocol provides a detailed description of experimental procedures, including Arabidopsis seedlings cultivation, the preparation of Agrobacterium suspensions, and subsequent steps leading to confocal microscope observation. Through this protocol, researchers can efficiently investigate gene function and subcellular localization in Arabidopsis cotyledons within 8 days in total.
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Arabidopsis , Plantones , Arabidopsis/genética , Arabidopsis/metabolismo , Plantones/genética , Plantones/metabolismo , Plantones/crecimiento & desarrollo , Vacio , Cotiledón/genética , Cotiledón/metabolismo , Transformación Genética , Expresión Génica , Plantas Modificadas Genéticamente/genética , Agrobacterium/genética , Regulación de la Expresión Génica de las Plantas , Microscopía ConfocalRESUMEN
The confocal laser scanning microscope allows the visualization of intracellular structures in greater detail than a widefield fluorescence microscope. Immunofluorescence (IF) techniques make use of the inherent ability of antibodies to bind to specific epitopes of specific proteins. Tagging these antibodies with an easily visualized molecule, e.g., a fluorophore, enables imaging in the fluorescence microscope. This is, however, a localization technique and will only give information about where certain proteins are; it does not provide the ultrastructural context provided by the transmission electron microscope. It also relies heavily on the accuracy and binding affinity of individual primary antibodies. Despite this, it is a commonly used, robust, and adaptable technique. In this chapter, we use a long-established IF protocol from our laboratory to locate EHDV proteins in a monolayer of infected cultured cells.