Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.490
Filtrar
1.
Methods Mol Biol ; 2203: 241-261, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833217

RESUMO

Coronavirus entry encompasses the initial steps of infection, from virion attachment to genome release. Advances in fluorescent labeling of viral and cellular components and confocal imaging enable broad spectrum studies on this process. Here, we describe methods for visualization of coronavirus entry into immortalized cell lines and 3D tissue culture models.


Assuntos
Coronavirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Microscopia Confocal/métodos , Linhagem Celular , Coronavirus/isolamento & purificação , Meios de Cultura/química , Endocitose , Humanos , Proteínas do Nucleocapsídeo/metabolismo , Ácidos Tri-Iodobenzoicos/química , Internalização do Vírus
2.
Dermatol Online J ; 26(3)2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32609438

RESUMO

Reflectance confocal microscopy (RCM) is a noninvasive real-time imaging technique that has been widely used for the diagnosis of skin cancer. More recently, it has been reported as a useful tool for the diagnosis and management of several inflammatory and infectious skin disorders. This article provides an overview of the current available applications of RCM use in cutaneous infections and infestations. PubMed was used to search the following terms in various combinations: reflectance confocal microscopy, skin, hair, nail, infection, parasitosis, mycosis, virus, bacteria. All papers were accordingly reviewed. In most cutaneous infections or infestations, the main alterations are found in the epidermis and upper dermis, where the accuracy of confocal microscopy is nearly similar to that of histopathology. The high resolution of this technique allows the visualization of most skin parasites, fungi, and a few bacteria. Although viruses cannot be identified because of their small size, viral cytopathic effects can be observed on keratinocytes. In addition, RCM can be used to monitor the response to treatment, thereby reducing unnecessary treatments.


Assuntos
Microscopia Confocal/métodos , Dermatopatias Infecciosas/diagnóstico por imagem , Dermatopatias Parasitárias/diagnóstico por imagem , Feminino , Humanos , Masculino , Pele/diagnóstico por imagem , Pele/microbiologia , Pele/parasitologia , Pele/patologia , Dermatopatias Infecciosas/patologia , Dermatopatias Parasitárias/patologia
3.
Angiology ; 71(10): 916-919, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32633543

RESUMO

Inflammation has a central role in atherosclerotic plaque formation and rupture. Intense macrophage inflammatory activity results in microcalcifications which are strongly associated with plaque vulnerability. Microcalcifications with specific critical size between 5 and 65 µ, located in the fibrous cap producing local mechanical stress on the plaque surface and may directly contribute to plaque rupture. Hence, accurate assessment of microcalcifications size and dimension has significant clinical importance. Current invasive and noninvasive plaque imaging has limited spatial resolution which limits accurate definition of microcalcifications in the atherosclerotic plaques. We describe a new imaging technique with high spatial resolution, based on confocal microscopic analysis, using a dedicated software which allows automatic characterization of microcalcifications and quantitative assessment of their extent and localization.


Assuntos
Calcinose/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Placa Aterosclerótica/diagnóstico por imagem , Humanos
4.
J Vis Exp ; (159)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32510475

RESUMO

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes' large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.


Assuntos
Divisão Celular/fisiologia , Drosophila/patogenicidade , Larva/patogenicidade , Microscopia Confocal/métodos , Pupa/patogenicidade , Testículo/metabolismo , Animais , Masculino , Testículo/citologia
5.
Food Chem ; 331: 127221, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32540697

RESUMO

Herein, a two-photon (TP) ratiometric fluorescent probe (NpFA) was developed for detecting formaldehyde (FA) in real food samples, living onion tissues and zebrafish by fluorescence resonance energy transfer (FRET) strategy. Specifically, a TP fluorophore as the donor and a FA turn-on naphthalimide fluorophore as the acceptor were connected by a non-conjugated linker to construct the TP-FRET-based NpFA, which exhibited a target-modulated ratiometric fluorescence response to FA rapidly with high selectivity and sensitivity during 65 s, and a large ratio ~5-fold enhancement at I550/I410 after addition of FA, displaying ~60-fold enhancement at 550 nm and a quite low DOC of 5.8 ± 0.2 nM. Moreover, NpFA has a good imaging resolution and depth of deep tissue penetration. Therefore, based on the above results, NpFA has the capability to be a useful tool for investigating FA in real samples application, and we also hope NpFA will further study of the physiological and pathological function of FA.


Assuntos
Corantes Fluorescentes/química , Análise de Alimentos/métodos , Formaldeído/análise , Microscopia Confocal/métodos , Imagem Óptica/métodos , Peixe-Zebra/metabolismo , Animais , Cromatografia em Camada Delgada , Transferência Ressonante de Energia de Fluorescência , Formaldeído/metabolismo , Células HeLa , Humanos
6.
Nat Commun ; 11(1): 2185, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366822

RESUMO

Signal amplification in biological systems is achieved by cooperatively recruiting multiple copies of regulatory biomolecules. Nevertheless, the multiplexing capability of artificial fluorescent amplifiers is limited due to the size limit and lack of modularity. Here, we develop Cayley tree-like fractal DNA frameworks to topologically encode the fluorescence states for multiplexed detection of low-abundance targets. Taking advantage of the self-similar topology of Cayley tree, we use only 16 DNA strands to construct n-node (n = 53) structures of up to 5 megadalton. The high level of degeneracy allows encoding 36 colours with 7 nodes by site-specifically anchoring of distinct fluorophores onto a structure. The fractal topology minimises fluorescence crosstalk and allows quantitative decoding of quantized fluorescence states. We demonstrate a spectrum of rigid-yet-flexible super-multiplex structures for encoded fluorescence detection of single-molecule recognition events and multiplexed discrimination of living cells. Thus, the topological engineering approach enriches the toolbox for high-throughput cell imaging.


Assuntos
DNA/química , Fluorescência , Fractais , Oligonucleotídeos/química , Algoritmos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia de Força Atômica/métodos , Microscopia Confocal/métodos , Nanoestruturas/química
7.
Nat Commun ; 11(1): 2184, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366843

RESUMO

Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane, yet how such proteins target and translocate to the nucleus remains in dispute. Most models propose transport through the nuclear pore complexes, but a central outstanding question is whether transit occurs through their central or peripheral channels. Using live-cell high-speed super-resolution single-molecule microscopy we could distinguish protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains containing nuclear localization signals into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels; however, the mutated proteins can still complete their translocation using only the peripheral channels, albeit at a reduced rate. Such proteins can still translocate using only the peripheral channels when central channel is blocked, but blocking the peripheral channels blocks translocation through both channels. This suggests that peripheral channel transport is the default mechanism that was adapted in evolution to include aspects of receptor-mediated central channel transport for directed trafficking of certain membrane proteins.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Recuperação de Fluorescência Após Fotodegradação , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte Proteico
8.
PLoS One ; 15(5): e0232912, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392236

RESUMO

The study of oral disease progression, in relation to the accumulation of subgingival biofilm in gingivitis and periodontitis is limited, due to either the ability to monitor plaque in vitro. When compared, optical spectroscopic techniques offer advantages over traditional destructive or biofilm staining approaches, making it a suitable alternative for the analysis and continued development of three-dimensional structures. In this work, we have developed a confocal Raman spectroscopy analysis approach towards in vitro subgingival plaque models. The main objective of this study was to develop a method for differentiating multiple oral subgingival bacterial species in planktonic and biofilm conditions, using confocal Raman microscopy. Five common subgingival bacteria (Fusobacterium nucleatum, Streptococcus mutans, Veillonella dispar, Actinomyces naeslundii and Prevotella nigrescens) were used and differentiated using a 2-way orthogonal Partial Least Square with Discriminant Analysis (O2PLS-DA) for the collected spectral data. In addition to planktonic growth, mono-species biofilms cultured using the 'Zürich Model' were also analyzed. The developed method was successfully used to predict planktonic and mono-species biofilm species in a cross validation setup. The results show differences in the presence and absence of chemical bands within the Raman spectra. The O2PLS-DA model was able to successfully predict 100% of all tested planktonic samples and 90% of all mono-species biofilm samples. Using this approach we have shown that Confocal Raman microscopy can analyse and predict the identity of planktonic and mono-species biofilm species, thus enabling its potential as a technique to map oral multi-species biofilm models.


Assuntos
Bactérias/isolamento & purificação , Gengivite/microbiologia , Microscopia Óptica não Linear/métodos , Periodontite/microbiologia , Actinomyces , Técnicas Bacteriológicas/métodos , Biofilmes/crescimento & desenvolvimento , Meios de Cultura , Fusobacterium nucleatum , Gengiva/microbiologia , Viabilidade Microbiana , Microbiota , Microscopia Confocal/métodos , Plâncton , Prevotella intermedia , Streptococcus mutans , Veillonella
9.
PLoS One ; 15(5): e0232847, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374768

RESUMO

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


Assuntos
Broncoscopia/métodos , Elastina/ultraestrutura , Doenças Pulmonares Intersticiais/patologia , Microscopia Confocal/métodos , Microscopia de Vídeo/métodos , Alvéolos Pulmonares/ultraestrutura , Idoso , Algoritmos , Automação , Sistemas Computacionais , Elastina/análise , Desenho de Equipamento , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Microscopia Confocal/instrumentação , Microscopia de Vídeo/instrumentação , Pessoa de Meia-Idade , não Fumantes , Alvéolos Pulmonares/química , Abandono do Hábito de Fumar , Aprendizado de Máquina Supervisionado
10.
Nat Methods ; 17(6): 636-642, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393832

RESUMO

Genetic screens using pooled CRISPR-based approaches are scalable and inexpensive, but restricted to standard readouts, including survival, proliferation and sortable markers. However, many biologically relevant cell states involve cellular and subcellular changes that are only accessible by microscopic visualization, and are currently impossible to screen with pooled methods. Here we combine pooled CRISPR-Cas9 screening with microraft array technology and high-content imaging to screen image-based phenotypes (CRaft-ID; CRISPR-based microRaft followed by guide RNA identification). By isolating microrafts that contain genetic clones harboring individual guide RNAs (gRNA), we identify RNA-binding proteins (RBPs) that influence the formation of stress granules, the punctate protein-RNA assemblies that form during stress. To automate hit identification, we developed a machine-learning model trained on nuclear morphology to remove unhealthy cells or imaging artifacts. In doing so, we identified and validated previously uncharacterized RBPs that modulate stress granule abundance, highlighting the applicability of our approach to facilitate image-based pooled CRISPR screens.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Microscopia Confocal/métodos , Estresse Oxidativo/genética , RNA Guia/genética , Proteínas de Ligação a RNA/genética , Análise Serial de Tecidos/métodos , Sistemas CRISPR-Cas/genética , Citoplasma/metabolismo , Humanos , Aprendizado de Máquina , Agregados Proteicos/genética
11.
Proc Natl Acad Sci U S A ; 117(22): 12352-12358, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32409609

RESUMO

Lutein and zeaxanthin are xanthophyll carotenoids that are highly concentrated in the human macula, where they protect the eye from oxidative damage and improve visual performance. Distinguishing lutein from zeaxanthin in images of the human retina in vivo or in donor eye tissues has been challenging because no available technology has been able to reliably differentiate between these two carotenoids, which differ only in the position of one C = C bond. Here, we report the differential distributions of lutein and zeaxanthin in human donor retinas mapped with confocal resonance Raman microscopy. Zeaxanthin is highly concentrated in the fovea, extending from the inner to the outer limiting membranes, with especially high concentrations in the outer plexiform layer, while lutein is much more diffuse at relatively lower concentration. Our results imply that zeaxanthin may play a more important role than lutein in human macular health and disease.


Assuntos
Luteína/análise , Retina/química , Zeaxantinas/análise , Humanos , Microscopia Confocal/métodos , Xantofilas/análise
12.
Muscle Nerve ; 62(1): 137-142, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32304246

RESUMO

BACKGROUND: Conventional processing of nerve for histomorphometry is resource-intensive, precluding use in intraoperative assessment of nerve quality during nerve transfer procedures. Stimulated Raman scattering (SRS) microscopy is a label-free technique that enables rapid and high-resolution histology. METHODS: Segments of healthy murine sciatic nerve, healthy human obturator nerve, and human cross-facial nerve autografts were imaged on a custom SRS microscope. Myelinated axon quantification was performed through segmentation using a random forest machine learning algorithm in commercial software. RESULTS: High contrast, high-resolution imaging of nerve morphology was obtained with SRS imaging. Automated myelinated axon quantification from cross-sections of healthy human nerve imaged using SRS was achieved. CONCLUSIONS: Herein, we demonstrate the use of a label-free technique for rapid imaging of murine and human peripheral nerve cryosections. We illustrate the potential of this technique to inform intraoperative decision-making through rapid automated quantification of myelinated axons using a machine learning algorithm.


Assuntos
Nervo Facial/química , Nervo Obturador/química , Nervo Isquiático/química , Análise Espectral Raman/métodos , Animais , Nervo Facial/anatomia & histologia , Humanos , Camundongos , Microscopia Confocal/métodos , Nervo Obturador/anatomia & histologia , Nervo Isquiático/anatomia & histologia
13.
Invest Ophthalmol Vis Sci ; 61(4): 39, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32340031

RESUMO

Purpose: The purpose of this study was to use three-dimensional confocal microscopy to quantify the spatial patterns of capillary network alterations in nonproliferative diabetic retinopathy (NPDR). Methods: The retinal microvasculature was perfusion-labelled in seven normal human donor eyes and six age-matched donor eyes with NPDR. The peripapillary microcirculation was studied using confocal scanning laser microscopy. Capillary density and diameters of the radial peripapillary capillary plexus (RPCP), superficial capillary plexus (SCP), intermediate capillary plexus (ICP), and deep capillary plexus (DCP) were quantified and compared. Three-dimensional visualization strategies were also used to compare the communications between capillary beds and precapillary arterioles and postcapillary venules. Results: Mean capillary diameter was significantly increased in the NPDR group (P < 0.001). Intercapillary distance was significantly increased in the DCP (P = 0.004) and RPCP (P = 0.022) of the NPDR group (P = 0.010) but not the SCP (P = 0.155) or ICP (P = 0.103). The NPDR group was associated with an increased frequency of inflow communication between the SCP and ICP/DCP and a decreased frequency of communication between the SCP and RPCP (P = 0.023). There was no difference in the patterns of outflow communications between the two groups (P = 0.771). Conclusions: This study demonstrates that capillary plexuses are nonuniformly perturbed in NPDR. These structural changes may be indicative of perturbations to blood flow patterns between different retinal layers. Our findings may aid the interpretation of previous clinical observations made using optical coherence tomography angiography as well as improve our understanding of the pathogenesis of NPDR.


Assuntos
Retinopatia Diabética/diagnóstico por imagem , Retinopatia Diabética/patologia , Imageamento Tridimensional , Vasos Retinianos/patologia , Austrália , Estudos de Casos e Controles , Enucleação Ocular , Feminino , Humanos , Técnicas In Vitro , Masculino , Microcirculação/fisiologia , Microscopia Confocal/métodos , Valores de Referência , Vasos Retinianos/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos
14.
Acta Diabetol ; 57(9): 1043-1047, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32246268

RESUMO

PURPOSE: To compare nonmydriatic montage widefield images with dilated fundus ophthalmoscopy for determining diabetic retinopathy (DR) severity. MATERIALS AND METHODS: In this prospective, observational, cross-sectional study, patients with a previous diagnosis of diabetes and without history of diabetes-associated ocular disease were screened for DR. Montage widefield imaging was obtained with a system that combines confocal technology with white-light emitting diode (LED) illumination (DRSplus, Centervue, Padua, Italy). Dilated fundus examination was performed by a retina specialist. RESULTS: Thirty-seven eyes (20 patients, 8 females) were finally included in the analysis. Mean age of the patients enrolled was 58.0 ± 11.6 years [range 31-80 years]. The level of DR identified on montage widefield images agreed exactly with indirect ophthalmoscopy in 97.3% (36) of eyes and was within 1 step in 100% (37) of eyes. Cohen's kappa coefficient (κ) was 0.96, this suggesting an almost perfect agreement between the two modalities in DR screening. Nonmydriatic montage widefield imaging acquisition time was significantly shorter than that of dilated clinical examination (p = 0.010). CONCLUSION: Nonmydriatic montage widefield images were compared favorably with dilated fundus examination in defining DR severity; however, they are acquired more rapidly.


Assuntos
Retinopatia Diabética/diagnóstico , Técnicas de Diagnóstico Oftalmológico , Programas de Rastreamento/métodos , Retina/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Dilatação , Feminino , Fundo de Olho , Humanos , Luz , Masculino , Microscopia Confocal/métodos , Pessoa de Meia-Idade , Midriáticos/farmacologia , Oftalmoscopia/métodos , Reconhecimento Automatizado de Padrão/métodos , Fotografação/métodos , Estudos Prospectivos , Retina/fisiopatologia
15.
PLoS One ; 15(4): e0231987, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320450

RESUMO

OBJECTIVES: Corneal nerve damage may be a surrogate marker for the risk of ischemic stroke. This study was undertaken to determine if there is greater corneal nerve damage in patients with recurrent ischemic stroke. METHODS: Corneal confocal microscopy (CCM) was used to quantify corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL) and corneal nerve fiber tortuosity (CNFT) in 31 patients with recurrent ischemic stroke, 165 patients with a first acute ischemic stroke and 23 healthy control subjects. RESULTS: Triglycerides (P = 0.004, P = 0.017), systolic BP (P = 0.000, P = 0.000), diastolic BP (P = 0.000, P = 0.000) and HbA1c (P = 0.000, P = 0.000) were significantly higher in patients with first and recurrent stroke compared to controls. There was no difference in age, BMI, HbA1c, total cholesterol, triglycerides, LDL, HDL, systolic and diastolic BP between patients with a first and recurrent ischemic stroke. However, CNFD was significantly lower (24.98±7.31 vs 29.07±7.58 vs 37.91±7.13, P<0.05) and CNFT was significantly higher (0.085±0.042 vs 0.064±0.037 vs 0.039±0.022, P<0.05) in patients with recurrent stroke compared to first stroke and healthy controls. CNBD (42.21±24.65 vs 50.46±27.68 vs 87.24±45.85, P<0.001) and CNFL (15.66±5.70, P<0.001 vs 17.38±5.06, P = 0.003) were equally reduced in patients with first and recurrent stroke compared to controls (22.72±5.14). CONCLUSIONS: Corneal confocal microscopy identified greater corneal nerve fibre loss in patients with recurrent stroke compared to patients with first stroke, despite comparable risk factors. Longitudinal studies are required to determine the prognostic utility of corneal nerve fiber loss in identifying patients at risk of recurrent ischemic stroke.


Assuntos
Isquemia Encefálica/complicações , Lesões da Córnea/diagnóstico por imagem , Microscopia Confocal/métodos , Acidente Vascular Cerebral/complicações , Adulto , Idoso , Isquemia Encefálica/fisiopatologia , Estudos de Casos e Controles , Lesões da Córnea/fisiopatologia , Hemoglobina A Glicada/análise , Humanos , Modelos Lineares , Pessoa de Meia-Idade , Nervo Oftálmico/diagnóstico por imagem , Nervo Oftálmico/fisiopatologia , Recidiva , Acidente Vascular Cerebral/fisiopatologia , Triglicerídeos/sangue
16.
Actas dermo-sifiliogr. (Ed. impr.) ; 111(3): 236-242, abr. 2020. ilus
Artigo em Espanhol | IBECS | ID: ibc-191526

RESUMO

La microscopia confocal ex vivo es un sistema de procesamiento de tejidos que permite el análisis histológico inmediato de tejidos extirpados por medio de láseres diodo con distintos espectros de longitud de onda. En estudios previos la microscopia confocal ex vivo reduce el tiempo de análisis de márgenes de forma muy notable, con una sensibilidad y especificidad respecto a la histopatología del 88% y 99%, respectivamente. Recientemente se ha desarrollado una nueva tecnología que es capaz de proveer imágenes digitales con mayor velocidad y mejor resolución que las obtenidas en los dispositivos previos. Mediante el método de fusión (láseres de fluorescencia y reflectancia que escanean simultáneamente), se reproduce con una tinción digital la hematoxilina y eosina de forma inmediata en cada imagen. La implementación de esta nueva tecnología ha abierto definitivamente una puerta en el diagnóstico inmediato de tejidos


The ex vivo confocal microscope is an imaging system designed to analyze freshly excised tissue using two diode lasers with different wavelengths. The technique can dramatically reduce margin analysis times and offers a sensitivity of 88% and a specificity of 89% relative to histopathology. A new technology has recently been developed that produces images more quickly and with a higher resolution than before. By means of a fusion mode that combines simultaneously scanned fluorescence and reflectance images, it produces digitally stained images that simulate the effect of hematoxylin-eosin staining. Application of this new technology has opened the door to real-time tissue diagnostics


Assuntos
Humanos , Microscopia Confocal/métodos , Pele/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/instrumentação , Microscopia Confocal/instrumentação , Fluorescência , Biópsia , Interpretação de Imagem Assistida por Computador/instrumentação , Carcinoma Basocelular/diagnóstico por imagem
18.
Am J Ophthalmol ; 217: 38-48, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32278770

RESUMO

PURPOSE: To correlate in vivo confocal microscopy morphologic features (IVCM-MF) and Acanthamoeba cyst density (ACD) with final best-corrected visual acuity (BCVA) in Acanthamoeba keratitis (AK). DESIGN: Retrospective cohort study. METHODS: Patient demographics, treatment outcome, and corresponding IVCM-MF performed at the acute stage of infection were analyzed. Inclusion criteria were microbiological positive AK cases seen at Moorfields Eye Hospital between February 2013 and October 2017. Statistical significance was assessed by multinomial regression and multiple linear regression analysis. Main outcome measure was final BCVA. RESULTS: A total of 157 eyes (157 patients) had AK. Absence of single-file round/ovoid objects was associated with a BCVA of 6/36 to 6/9 (odds ratio [OR] 8.13; 95% confidence interval [CI], 1.55-42.56, P = .013) and ≥6/6 (OR 10.50; 95% CI, 2.12-51.92, P = .004) when compared to no perception of light to 6/60. Absence of rod/spindle objects was associated with a BCVA of ≥6/6 (OR 4.55; 95% CI, 1.01-20.45, P = .048). Deep stromal/ring infiltrate was associated with single-file round/ovoid objects (OR 7.78; 95% CI, 2.69-22.35, P < .001), rod/spindle objects (OR 7.05; 95% CI, 2.11-23.59, P = .002), and binary round/ovoid objects (OR 3.45; 95% CI, 1.17-10.14, P = .024). There was a positive association between ACD and treatment duration (ß = 0.14, P = .049), number of IVCM-MF (ß = 0.34, P = .021), and clusters of round/ovoid objects (ß = 0.29, P = .002). CONCLUSIONS: Specific IVCM-MF correlate with ACD and clinical staging of disease, and are prognostic indicators for a poorer visual outcome.


Assuntos
Ceratite por Acanthamoeba/diagnóstico , Córnea/patologia , Infecções Oculares Fúngicas/diagnóstico , Microscopia Confocal/métodos , Acuidade Visual , Acanthamoeba/genética , Ceratite por Acanthamoeba/microbiologia , Ceratite por Acanthamoeba/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Córnea/microbiologia , DNA Fúngico/análise , Infecções Oculares Fúngicas/microbiologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Adulto Jovem
19.
PLoS Negl Trop Dis ; 14(3): e0008007, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196491

RESUMO

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge.


Assuntos
Doença de Chagas/parasitologia , Replicação do DNA , Estágios do Ciclo de Vida , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Feminino , Genes Reporter , Microscopia Intravital/métodos , Camundongos SCID , Microscopia Confocal/métodos , Coloração e Rotulagem/métodos , Trypanosoma cruzi/genética
20.
Arq Bras Oftalmol ; 83(2): 146-148, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32159595

RESUMO

Lisch corneal dystrophy is a rare corneal disease characterized by the distinctive feature of highly vacuolated cells. Although this feature is important, the nature of these vacuoles within corneal cells remains unknown. Here, we sought to analyze corneal cells from a patient diagnosed with Lisch dystrophy to characterize the vacuoles within these cells. Analyses using histopathology examination, confocal microscopy, and transmission electron microscopy were all consistent with previous descriptions of Lisch cells. Importantly, the vacuoles within these cells appeared to be autophagosomes and autolysosomes, and could be stained with an anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) antibody. Taken together, these findings indicate that the vacuoles we observed within superficial corneal cells of a patient with Lisch corneal dystrophy constituted autophagosomes and autolysosomes; this finding has not been previously reported and suggests a need for further analyses to define the role of autophagy in this ocular disease.


Assuntos
Autofagossomos/patologia , Distrofias Hereditárias da Córnea/patologia , Vacúolos/patologia , Adulto , Distrofias Hereditárias da Córnea/diagnóstico por imagem , Opacidade da Córnea/diagnóstico por imagem , Opacidade da Córnea/patologia , Feminino , Humanos , Microautofagia , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , Tomografia de Coerência Óptica/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA