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Purpose: Raman spectroscopy allows molecular changes to be quantified in vivo from the tissues like the retina. Here we aimed to assess the metabolic changes in the retina of patients with multiple sclerosis (MS). Methods: We built a Raman spectroscopy prototype by connecting a scanning laser ophthalmoscope to a spectrophotometer. We defined the spectra of 10 molecules participating on energy supply, axon biology, or synaptic damage, which have been shown to be altered in the brain of patients with MS: cytochrome C, flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NADH), N-acetyl-aspartate (NAA), excitotoxicity, glutamate, amyloid ß (Aß), τ and α-synuclein (SNCA), phosphatidyl-ethanolamine, and phosphatidyl-choline. We studied these molecules in a prospective cohort of patients with MS, either in the chronic phase or during relapses of acute optic neuritis (AON). Results: Significant changes to all these molecules were associated with age in healthy individuals. There was a significant decrease in NADH and a trend toward a decrease in NAA in patients with MS, as well as an increase in Aß compared with healthy controls. Moreover, NADH and FAD increased over time in a longitudinal analysis of patients with MS, whereas Aß diminished. In patients with acute retinal inflammation due to AON, there was a significant increase in FAD and a decrease in SNCA in the affected retina. Moreover, glutamate levels increased in the affected eyes after a 6-month follow-up. Conclusions: Alterations of molecules related to axonal degeneration are observed during neuroinflammation and show dynamic changes over time, suggesting progressive neurodegeneration.
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Biomarcadores/metabolismo , Proteínas do Olho/metabolismo , Esclerose Múltipla/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doenças Retinianas/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Análise Espectral Raman , Tomografia de Coerência ÓpticaRESUMO
PURPOSE: To test whether graph analysis of vascular images obtained with hepatic dynamic contrast material-enhanced (DCE) ultrasonography (US) allows calculation of the degree of organization of the liver circulation and whether graph properties are correlated to the severity of portal hypertension. MATERIALS AND METHODS: Institutional review board approval and written informed consent were obtained. Fifteen patients with liver cirrhosis (nine men; mean age ± standard deviation, 55 years ± 8) who underwent DCE US and hepatic venous pressure gradient (HVPG) measurement and four healthy subjects (two men and two women; mean age, 34 years ± 4) were included between January 2007 and December 2008. Individual graph models ("vascular connectomes") were computed on the basis of time series analysis of video sequences of DCE US examinations (conducted with the disruption-reperfusion technique). Graph analysis was performed, and the clustering coefficient C was calculated. Correlations between clustering coefficient and HVPG were assessed. RESULTS: Healthy subjects had a high clustering coefficient of vascular connectome (C = 0.4447; interquartile range [IQR], 0.3864-0.4679), suggesting a highly organized hepatic vascular network. Conversely, patients with cirrhosis showed a low clustering coefficient, indicating disruption of normal anatomy (C = 0.0288; IQR, 0.0157-0.0861; P = .001 vs healthy subjects). The clustering coefficient decreased as HVPG increased, with a clustering coefficient of 0.0237 (IQR, 0.0066-0.0378) in patients with HVPG of at least 10 mm Hg versus 0.1180 (IQR, 0.0987-0.1414) in those with HVPG of less than 10 mm Hg (P = .006). The correlation between the best model derived from the distribution of the clustering coefficient (10 bins) of vascular connectome and HVPG had a Pearson correlation of 0.977 (root mean squared error, 1.57 mm Hg; P < .0001). CONCLUSION: This pilot study demonstrates that graph modeling of vascular connectivity based on video processing of liver DCE US examinations and subsequent graph analysis enable calculation of personalized parameters that reflect the degree of organization of the hepatic microvascular network and are correlated to the severity of portal hypertension in cirrhosis.
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Hipertensão Portal/diagnóstico por imagem , Fígado/irrigação sanguínea , Adulto , Idoso , Meios de Contraste , Feminino , Humanos , Hipertensão Portal/complicações , Cirrose Hepática/complicações , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Índice de Gravidade de Doença , Ultrassonografia/métodosRESUMO
BACKGROUND: Intrauterine growth restriction (IUGR) induces fetal cardiac remodelling and dysfunction, which persists postnatally and may explain the link between low birth weight and increased cardiovascular mortality in adulthood. However, the cellular and molecular bases for these changes are still not well understood. We tested the hypothesis that IUGR is associated with structural and functional gene expression changes in the fetal sarcomere cytoarchitecture, which remain present in adulthood. METHODS AND RESULTS: IUGR was induced in New Zealand pregnant rabbits by selective ligation of the utero-placental vessels. Fetal echocardiography demonstrated more globular hearts and signs of cardiac dysfunction in IUGR. Second harmonic generation microscopy (SHGM) showed shorter sarcomere length and shorter A-band and thick-thin filament interaction lengths, that were already present in utero and persisted at 70 postnatal days (adulthood). Sarcomeric M-band (GO: 0031430) functional term was over-represented in IUGR fetal hearts. CONCLUSION: The results suggest that IUGR induces cardiac dysfunction and permanent changes on the sarcomere.
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Modelos Animais de Doenças , Retardo do Crescimento Fetal/fisiopatologia , Coração Fetal/fisiopatologia , Feto/fisiopatologia , Sarcômeros/diagnóstico por imagem , Animais , Biomarcadores/análise , Pressão Sanguínea , Peso Corporal , Ecocardiografia , Feminino , Retardo do Crescimento Fetal/diagnóstico por imagem , Coração Fetal/diagnóstico por imagem , Perfilação da Expressão Gênica , Tamanho do Órgão , Placenta/metabolismo , Gravidez , CoelhosRESUMO
The identification of biomarkers of vascular cognitive impairment is urgent for its early diagnosis. The aim of this study was to detect and monitor changes in brain structure and connectivity, and to correlate them with the decline in executive function. We examined the feasibility of early diagnostic magnetic resonance imaging (MRI) to predict cognitive impairment before onset in an animal model of chronic hypertension: Spontaneously Hypertensive Rats. Cognitive performance was tested in an operant conditioning paradigm that evaluated learning, memory, and behavioral flexibility skills. Behavioral tests were coupled with longitudinal diffusion weighted imaging acquired with 126 diffusion gradient directions and 0.3 mm(3) isometric resolution at 10, 14, 18, 22, 26, and 40 weeks after birth. Diffusion weighted imaging was analyzed in two different ways, by regional characterization of diffusion tensor imaging (DTI) indices, and by assessing changes in structural brain network organization based on Q-Ball tractography. Already at the first evaluated times, DTI scalar maps revealed significant differences in many regions, suggesting loss of integrity in white and gray matter of spontaneously hypertensive rats when compared to normotensive control rats. In addition, graph theory analysis of the structural brain network demonstrated a significant decrease of hierarchical modularity, global and local efficacy, with predictive value as shown by regional three-fold cross validation study. Moreover, these decreases were significantly correlated with the behavioral performance deficits observed at subsequent time points, suggesting that the diffusion weighted imaging and connectivity studies can unravel neuroimaging alterations even overt signs of cognitive impairment become apparent.
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The purpose of the study was to evaluate the association between a quantitative texture analysis of early neonatal brain ultrasound images and later neurobehavior in preterm infants. A prospective cohort study including 120 preterm (<33 wk of gestational age) infants was performed. Cranial ultrasound images taken early after birth were analyzed in six regions of interest using software based on texture analysis. The resulting texture scores were correlated with the Neonatal Behavioural Assessment Scale (NBAS) at term-equivalent age. The ability of texture scores, in combination with clinical data and standard ultrasound findings, to predict the NBAS results was evaluated. Texture scores were significantly associated with all but one NBAS domain and better predicted NBAS results than clinical data and standard ultrasound findings. The best predictive value was obtained by combining texture scores with clinical information and ultrasound standard findings (area under the curve = 0.94). We conclude that texture analysis of neonatal cranial ultrasound-extracted quantitative features that correlate with later neurobehavior has a higher predictive value than the combination of clinical data with abnormalities in conventional cranial ultrasound.
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Mapeamento Encefálico/métodos , Desenvolvimento Infantil/fisiologia , Ecoencefalografia/métodos , Processamento de Imagem Assistida por Computador/métodos , Comportamento do Lactente/fisiologia , Análise de Variância , Estudos de Coortes , Feminino , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Estudos ProspectivosRESUMO
Automatic quantification of cardiac muscle properties in tissue sections might provide important information related to different types of diseases. Second harmonic generation (SHG) imaging provides a stain-free microscopy approach to image cardiac fibers that, combined with our methodology of the automated measurement of the ultrastructure of muscle fibers, computes a reliable set of quantitative image features (sarcomere length, A-band length, thick-thin interaction length, and fiber orientation). We evaluated the performance of our methodology in computer-generated muscle fibers modeling some artifacts that are present during the image acquisition. Then, we also evaluated it by comparing it to manual measurements in SHG images from cardiac tissue of fetal and adult rabbits. The results showed a good performance of our methodology at high signal-to-noise ratio of 20 dB. We conclude that our automated measurements enable reliable characterization of cardiac fiber tissues to systematically study cardiac tissue in a wide range of conditions.
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Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Técnicas Fotoacústicas/métodos , Sarcômeros/química , Algoritmos , Animais , Simulação por Computador , Miocárdio/citologia , Coelhos , Razão Sinal-RuídoRESUMO
OBJECTIVE: The purpose of this study was to assess whether abnormal cardiac function in human fetuses with intrauterine growth restriction (IUGR) is associated with ultrastructural differences in the cardiomyocyte sarcomere. STUDY DESIGN: Nine severe early-onset IUGR fetuses and 9 normally grown fetuses (appropriate growth for gestational age) who died in the perinatal period were included prospectively. Cardiac function was assessed by echocardiography and levels of B-type natriuretic peptide and troponin-I. Heart sections were imaged by second harmonic generation microscopy, which allowed unstained visualization of cardiomyocyte's sarcomere length. RESULTS: Echocardiographic and biochemical markers showed signs of severe cardiac dysfunction in IUGR fetuses. Second harmonic generation microscopy demonstrated a significantly shorter sarcomere length in IUGR as compared with appropriate growth for gestational age fetuses. CONCLUSION: IUGR is associated with changes in the cardiomyocyte contractile machinery in the form of shorter sarcomere length, which could help to explain the cardiac dysfunction previously documented in IUGR.
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Retardo do Crescimento Fetal/fisiopatologia , Feto/fisiopatologia , Miócitos Cardíacos/ultraestrutura , Peptídeo Natriurético Encefálico/análise , Sarcômeros/ultraestrutura , Troponina I/análise , Estudos de Casos e Controles , Ecocardiografia , Feminino , Retardo do Crescimento Fetal/diagnóstico por imagem , Retardo do Crescimento Fetal/patologia , Idade Gestacional , Humanos , Gravidez , Ultrassonografia Pré-NatalRESUMO
Retinal tissue is damaged during inflammation in Multiple Sclerosis. We assessed molecular changes in inflamed murine retinal cultures by Raman spectroscopy. Partial Least Squares-Discriminant analysis (PLS-DA) was able to classify retina cultures as inflamed with high accuracy. Using Multivariate Curve Resolution (MCR) analysis, we deconvolved 6 molecular components suffering dynamic changes along inflammatory process. Those include the increase of immune mediators (Lipoxygenase, iNOS and TNFα), changes in molecules involved in energy production (Cytochrome C, phenylalanine and NADH/NAD+) and decrease of Phosphatidylcholine. Raman spectroscopy combined with multivariate analysis allows monitoring the evolution of retina inflammation.
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Imagem Molecular , Doenças Retinianas/patologia , Análise Espectral Raman , Animais , Células Cultivadas , Análise Discriminante , Metabolismo Energético , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Análise dos Mínimos Quadrados , Metabolismo dos Lipídeos , Camundongos , Análise Multivariada , Doenças Retinianas/imunologia , Doenças Retinianas/metabolismo , Células Ganglionares da Retina/patologia , Fatores de TempoRESUMO
OBJECTIVE: The objective of the study was to evaluate the performance of automatic quantitative ultrasound analysis (AQUA) texture extractor to predict fetal lung maturity tests in amniotic fluid. STUDY DESIGN: Singleton pregnancies (24.0-41.0 weeks) undergoing amniocentesis to assess fetal lung maturity (TDx fetal lung maturity assay [FLM]) were included. A manual-delineated box was placed in the lung area of a 4-chamber view of the fetal thorax. AQUA transformed the information into a set of descriptors. Genetic algorithms extracted the most relevant descriptors and then created and validated a model that could distinguish between mature or immature fetal lungs using TDx-FLM as a reference. RESULTS: Gestational age at enrollment was (mean [SD]) 32.2 (4.5) weeks. According to the TDx-FLM results, 41 samples were mature and 62 were not. The imaging biomarker based on AQUA presented a sensitivity 95.1%, specificity 85.7%, and an accuracy 90.3% to predict a mature or immature lung. CONCLUSION: Fetal lung ultrasound textures extracted by AQUA provided robust features to predict TDx-FLM results.
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Maturidade dos Órgãos Fetais , Pulmão/diagnóstico por imagem , Ultrassonografia Pré-Natal/métodos , Adulto , Amniocentese , Líquido Amniótico , Feminino , Idade Gestacional , Humanos , Valor Preditivo dos Testes , Gravidez , Sensibilidade e EspecificidadeRESUMO
Based on its polarization dependency, second harmonic generation (PSHG) microscopy has been proven capable to structurally characterize molecular architectures in different biological samples. By exploiting this polarization dependency of the SHG signal in every pixel of the image, average quantitative structural information can be retrieved in the form of PSHG image histograms. In the present study we experimentally show how the PSHG image histograms can be affected by the organization of the SHG active molecules. Our experimental scenario grounds on two inherent properties of starch granules. Firstly, we take advantage of the radial organization of amylopectin molecules (the SHG source in starch) to attribute shifts of the image histograms to the existence of tilted off the plane molecules. Secondly, we use the property of starch to organize upon hydration to demonstrate that the degree of structural order at the molecular level affects the width of the PSHG image histograms. The shorter the width is the more organized the molecules in the sample are, resulting in a reliable method to measure order. The implication of this finding is crucial to the interpretation of PSHG images used for example in tissue diagnostics.
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OBJECTIVE: To evaluate the feasibility and reproducibility of fetal lung texture analysis using a novel automatic quantitative ultrasound analysis and to assess its correlation with gestational age. METHODS: Prospective cross-sectional observational study. To evaluate texture features, 957 left and right lung images in a 2D four-cardiac-chamber view plane were previously delineated from fetuses between 20 and 41 weeks of gestation. Quantification of lung texture was performed by the Automatic Quantitative Ultrasound Analysis (AQUA) software to extract image features. A standard learning approach composed of feature transformation and a regression model was used to evaluate the association between texture features and gestational age. RESULTS: The association between weeks of gestation and fetal lung texture quantified by the AQUA software presented a Pearson correlation of 0.97. The association was not influenced by delineation parameters such as region of interest (ROI) localization, ROI size, right/left lung selected or sonographic parameters such as ultrasound equipment or transducer used. CONCLUSIONS: Fetal lung texture analysis measured by the AQUA software demonstrated a strong correlation with gestational age. This supports further research to explore the use of this technology to the noninvasive prediction of fetal lung maturity.
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Idade Gestacional , Pulmão/diagnóstico por imagem , Pulmão/embriologia , Software , Ultrassonografia Pré-Natal/métodos , Adulto , Estudos Transversais , Estudos de Viabilidade , Feminino , Humanos , Gravidez , Estudos Prospectivos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Intrauterine growth restriction (IUGR) affects 5-10% of all newborns and is associated with a high risk of abnormal neurodevelopment. The timing and patterns of brain reorganization underlying IUGR are poorly documented. We developed a rabbit model of IUGR allowing neonatal neurobehavioral assessment and high resolution brain diffusion magnetic resonance imaging (MRI). The aim of the study was to describe the pattern and functional correlates of fetal brain reorganization induced by IUGR. METHODOLOGY/PRINCIPAL FINDINGS: IUGR was induced in 10 New Zealand fetal rabbits by ligation of 40-50% of uteroplacental vessels in one horn at 25 days of gestation. Ten contralateral horn fetuses were used as controls. Cesarean section was performed at 30 days (term 31 days). At postnatal day +1, neonates were assessed by validated neurobehavioral tests including evaluation of tone, spontaneous locomotion, reflex motor activity, motor responses to olfactory stimuli, and coordination of suck and swallow. Subsequently, brains were collected and fixed and MRI was performed using a high resolution acquisition scheme. Global and regional (manual delineation and voxel based analysis) diffusion tensor imaging parameters were analyzed. IUGR was associated with significantly poorer neurobehavioral performance in most domains. Voxel based analysis revealed fractional anisotropy (FA) differences in multiple brain regions of gray and white matter, including frontal, insular, occipital and temporal cortex, hippocampus, putamen, thalamus, claustrum, medial septal nucleus, anterior commissure, internal capsule, fimbria of hippocampus, medial lemniscus and olfactory tract. Regional FA changes were correlated with poorer outcome in neurobehavioral tests. CONCLUSIONS: IUGR is associated with a complex pattern of brain reorganization already at birth, which may open opportunities for early intervention. Diffusion MRI can offer suitable imaging biomarkers to characterize and monitor brain reorganization due to fetal diseases.
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Comportamento Animal , Encéfalo/patologia , Imagem de Difusão por Ressonância Magnética , Retardo do Crescimento Fetal/fisiopatologia , Animais , Animais Recém-Nascidos , Modelos Animais , Neuropsicologia , CoelhosRESUMO
Intrauterine growth restriction (IUGR) due to placental insufficiency affects 5-10% of all pregnancies and it is associated with a wide range of short- and long-term neurodevelopmental disorders. Prediction of neurodevelopmental outcomes in IUGR is among the clinical challenges of modern fetal medicine and pediatrics. In recent years several studies have used magnetic resonance imaging (MRI) to demonstrate differences in brain structure in IUGR subjects, but the ability to use MRI for individual predictive purposes in IUGR is limited. Recent research suggests that MRI in vivo access to brain connectivity might have the potential to help understanding cognitive and neurodevelopment processes. Specifically, MRI based connectomics is an emerging approach to extract information from MRI data that exhaustively maps inter-regional connectivity within the brain to build a graph model of its neural circuitry known as brain network. In the present study we used diffusion MRI based connectomics to obtain structural brain networks of a prospective cohort of one year old infants (32 controls and 24 IUGR) and analyze the existence of quantifiable brain reorganization of white matter circuitry in IUGR group by means of global and regional graph theory features of brain networks. Based on global and regional analyses of the brain network topology we demonstrated brain reorganization in IUGR infants at one year of age. Specifically, IUGR infants presented decreased global and local weighted efficiency, and a pattern of altered regional graph theory features. By means of binomial logistic regression, we also demonstrated that connectivity measures were associated with abnormal performance in later neurodevelopmental outcome as measured by Bayley Scale for Infant and Toddler Development, Third edition (BSID-III) at two years of age. These findings show the potential of diffusion MRI based connectomics and graph theory based network characteristics for estimating differences in the architecture of neural circuitry and developing imaging biomarkers of poor neurodevelopment outcome in infants with prenatal diseases.
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Mapeamento Encefálico/métodos , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Desenvolvimento Infantil , Retardo do Crescimento Fetal/patologia , Retardo do Crescimento Fetal/fisiopatologia , Imageamento por Ressonância Magnética , Rede Nervosa/patologia , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Estudos ProspectivosRESUMO
OBJECTIVES: Diagnosis of white matter damage by cranial ultrasound imaging is still subject to interobserver variability and has limited sensitivity for predicting abnormal neurodevelopment later in life. In this study, we evaluated the ability of a semiautomated method based on ultrasound texture analysis to identify patterns that correlate with the ultrasound diagnosis of white matter damage. METHODS: The study included 44 very preterm neonates born at a median gestational age of 29 weeks 3 days (range, 26 weeks-31 weeks 6 days). Patients underwent cranial ultrasound scans within 1 week of birth and between 14 and 31 days of life. Periventricular leukomalacia was diagnosed by experienced clinicians on the 14- to 31-day scan according to standard criteria. To perform the texture analysis, 4 regions of interest were delineated in stored images: left and right periventricular areas and choroid plexuses. A classification algorithm was developed on the basis of the best combination of texture coefficients to correlate with the clinical diagnosis, and the ability of this algorithm to predict a later diagnosis of periventricular leukomalacia on the first scan was evaluated using a leave-one-out cross-validation. RESULTS: Periventricular leukomalacia was diagnosed by the standard procedure in 14 of 44 neonates. The texture classification algorithm performed on the first scan could identify cases with a later diagnosis of periventricular leukomalacia with sensitivity of 100% and accuracy of 97.7%. CONCLUSIONS: These data support the notion that semiautomated quantitative ultrasound analysis achieves early identification of changes in subclinical stages and warrant further investigation of the role of ultrasound texture analysis methods to improve early detection of neonatal brain damage.
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Ecoencefalografia/métodos , Recém-Nascido Prematuro , Leucomalácia Periventricular/diagnóstico por imagem , Fibras Nervosas Mielinizadas/diagnóstico por imagem , Reconhecimento Automatizado de Padrão , Algoritmos , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Masculino , Estudos Prospectivos , Sensibilidade e Especificidade , Estatísticas não ParamétricasRESUMO
In isolated congenital diaphragmatic hernia, prenatal prediction is made based on measurements of lung size and the presence of liver herniation into the thorax. A subset of fetuses likely to die in the postnatal period is eligible for fetal intervention that can promote lung growth. Rather than anatomical repair, this is now attempted by temporary fetal endoscopic tracheal occlusion (FETO). Herein we describe purpose-designed instruments that were developed thanks to a grant from the European Commission. The feasibility and safety of FETO have now been demonstrated in several active fetal surgery programs. The most frequent complication of the procedure is preterm premature rupture of the membranes, which is probably iatrogenic in nature. It does have an impact on gestational age at delivery and complicates balloon removal. FETO is associated with an apparent increase in survival compared with same severity controls, although this needs to be evaluated in a formal trial. The time has come to do so.
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Fetoscopia/métodos , Traqueia/cirurgia , Oclusão com Balão/instrumentação , Oclusão com Balão/métodos , Cateterismo/instrumentação , Cateterismo/métodos , Desenho de Equipamento/métodos , Feminino , Fetoscopia/instrumentação , Feto/cirurgia , Idade Gestacional , Hérnia Diafragmática/diagnóstico por imagem , Hérnia Diafragmática/mortalidade , Hérnia Diafragmática/cirurgia , Hérnias Diafragmáticas Congênitas , Humanos , Lactente , Recém-Nascido , Gravidez , Resultado da Gravidez , Instrumentos Cirúrgicos , Taxa de Sobrevida , Traqueia/embriologia , Resultado do Tratamento , Ultrassonografia Pré-NatalRESUMO
Pixel resolution polarization-sensitive second harmonic generation (PSHG) imaging has been recently shown as a promising imaging modality, by largely enhancing the capabilities of conventional intensity-based SHG microscopy. PSHG is able to obtain structural information from the elementary SHG active structures, which play an important role in many biological processes. Although the technique is of major interest, acquiring such information requires long offline processing, even with current computers. In this paper, we present an approach based on Fourier analysis of the anisotropy signature that allows processing the PSHG images in less than a second in standard single core computers. This represents a temporal improvement of several orders of magnitude compared to conventional fitting algorithms. This opens up the possibility for fast PSHG information with the subsequent benefit of potential use in medical applications.
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Análise de Fourier , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Polarização/métodos , AnisotropiaRESUMO
In this work we show that a pulsed laser light placed at a distance is able to modulate the growth of axons of primary neuronal cell cultures. In our experiments continuous wave (CW), chopped CW and modelocked fs (FS) laser light was focused through a microscope objective to a point placed at a distance of about 15 microm from the growth cone. We found that CW light does not produce any significant influence on the axon growth. In contrast, when using pulsed light (chopped CW light or FS pulses), the beam was able to modify the trajectory of the axons, attracting approximately 45% of the observed cases to the beam spot. Such effect could possibly indicate the capacity of neurons to interpret the pulsating NIR light as the source of other nearby cells, resulting in extension of processes in the direction of the source.
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Axônios/efeitos da radiação , Crescimento Celular/efeitos da radiação , Cones de Crescimento/efeitos da radiação , Lasers , Luz , Neurônios/efeitos da radiação , Animais , Axônios/fisiologia , Células Cultivadas , Córtex Cerebral/fisiologia , Córtex Cerebral/efeitos da radiação , Cones de Crescimento/fisiologia , Camundongos , Camundongos Endogâmicos , Neurônios/fisiologia , Pressão , Gravação em VídeoRESUMO
In this paper we provide, for the first time to our knowledge, the effective orientation of the SHG source in cultured cortical neuronal processes in vitro. This is done by the use of the polarization sensitive second harmonic generation (PSHG) imaging microscopy technique. By performing a pixel-level resolution analysis we found that the SHG dipole source has a distribution of angles centered at thetae =33.96 degrees , with a bandwidth of Deltathetae = 12.85 degrees . This orientation can be related with the molecular geometry of the tubulin heterodimmer contained in microtubules.
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Córtex Cerebral/citologia , Aumento da Imagem/instrumentação , Microscopia de Fluorescência/instrumentação , Neurônios/citologia , Dispositivos Ópticos , Animais , Simulação por Computador , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Aumento da Imagem/métodos , Luz , Microscopia de Fluorescência/métodos , Modelos Teóricos , Ratos , Ratos Sprague-Dawley , Espalhamento de RadiaçãoRESUMO
In this study, the second harmonic generation (SHG) response to polarization and subsequent data analysis is used to discriminate, in the same image, different SHG source architectures with pixel resolution. This is demonstrated in a mammalian tissue containing both skeletal muscle and fibrilar collagen. The SHG intensity variation with the input polarization (PSHG) is fitted pixel by pixel in the image using an algorithm based on a generalized biophysical model. The analysis provides the effective orientation, theta(e), of the different SHG active structures (harmonophores) at every pixel. This results in a new image in which collagen and muscle are clearly differentiated. In order to quantify the SHG response, the distribution of theta(e) for every harmonophore is obtained. We found that for collagen, the distribution was centered at theta(e) = 42.7 degrees with a full width at half maximum of theta = 5.9 degrees while for muscle theta(e) = 65.3 degrees , with theta = 7.7 degrees . By comparing these distributions, a quantitative measurement of the discrimination procedure is provided.
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Algoritmos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Polarização/métodos , Músculo Esquelético/citologia , Refratometria/métodos , Animais , Masculino , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The polarization dependence of second harmonic generation (SHG) microscopy is used to uncover structural information in different muscle cells in a living Caenorhabditis elegans (C. elegans) nematode. This is done by using a generalized biophysical model in which element ratios for the associated second-order nonlinear tensor and angular orientations for thick filaments are retrieved using a pixel-by-pixel fitting algorithm. As a result, multiple arbitrary orientations of thick filaments, at the pixel-resolution level, are revealed in the same image. The validity of our method is first corroborated in well-organized thick filaments such as the nonfibrilar body wall muscles. Next, a region of the nonstriated muscular cells of the pharynx is analyzed by showing different regions with homogenous orientations of thick filament as well as their radial distribution. As a result, different sets of the nonstriated muscle cell groups in the pharynx of this nematode were exposed. This methodology is presented as a filtering mechanism to uncover biological information unreachable by common intensity SHG microscopy. Finally, a method to experimentally retrieve the distribution of the effective orientation of active SHG molecules is proposed and tested.