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1.
Nat Commun ; 15(1): 27, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-38167277

RESUMO

Direct interactions between receptors at the neuronal surface have long been proposed to tune signaling cascades and neuronal communication in health and disease. Yet, the lack of direct investigation methods to measure, in live neurons, the interaction between different membrane receptors at the single molecule level has raised unanswered questions on the biophysical properties and biological roles of such receptor interactome. Using a multidimensional spectral single molecule-localization microscopy (MS-SMLM) approach, we monitored the interaction between two membrane receptors, i.e. glutamatergic NMDA (NMDAR) and G protein-coupled dopamine D1 (D1R) receptors. The transient interaction was randomly observed along the dendritic tree of hippocampal neurons. It was higher early in development, promoting the formation of NMDAR-D1R complexes in an mGluR5- and CK1-dependent manner, favoring NMDAR clusters and synaptogenesis in a dopamine receptor signaling-independent manner. Preventing the interaction in the neonate, and not adult, brain alters in vivo spontaneous neuronal network activity pattern in male mice. Thus, a weak and transient interaction between NMDAR and D1R plays a structural and functional role in the developing brain.


Assuntos
N-Metilaspartato , Receptores de Dopamina D1 , Camundongos , Animais , Receptores de Dopamina D1/metabolismo , Transdução de Sinais/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Neurônios/metabolismo
2.
Front Bioinform ; 2: 813494, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36304321

RESUMO

Single molecule localization (SML) and tracking (SPT) techniques, such as (spt)PALM, (u/DNA)PAINT and quantum dot tracking, have given unprecedented insight into the nanoscale molecular organization and dynamics in living cells. They allow monitoring individual proteins with millisecond temporal resolution and high spatial resolution (<30 nm) by precisely localizing the point spread function (PSF) of individual emitters and tracking their position over time. While SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups are restricted in the number of proteins they can probe simultaneously and usually have to tradeoff between the number of colors, the spatio-temporal resolution, and the field of view. Yet, localizing and tracking several proteins simultaneously at high spatial and temporal resolution within large field of views can provide important biological insights. By employing a dual-objective spectral imaging configuration compatible with live cell imaging combined with dedicated computation tools, we demonstrate simultaneous 3D single particle localization and tracking of multiple distinct species over large field of views to be feasible without compromising spatio-temporal resolution. The dispersive element introduced into the second optical path induces a spectrally dependent displacement, which we used to analytically separate up to five different fluorescent species of single emitters based on their emission spectra. We used commercially available microscope bodies aligned one on top of the other, offering biologists with a very ergonomic and flexible instrument covering a broad range of SMLM applications. Finally, we developed a powerful freely available software, called PALMTracer, which allows to quantitatively assess 3D + t + λ SMLM data. We illustrate the capacity of our approach by performing multi-color 3D DNA-PAINT of fixed samples, and demonstrate simultaneous tracking of multiple receptors in live fibroblast and neuron cultures.

3.
Biol Cell ; 113(12): 492-506, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34498765

RESUMO

Leucine Rich Repeat Transmembrane proteins (LRRTMs) are neuronal cell adhesion molecules involved in synapse development and plasticity. LRRTM2 is the most synaptogenic isoform of the family, and its expression is strongly restricted to excitatory synapses in mature neurons. However, the mechanisms by which LRRTM2 is trafficked and stabilized at synapses remain unknown. Here, we examine the role of LRRTM2 intracellular domain on its membrane expression and stabilization at excitatory synapses, using a knock-down strategy combined to single molecule tracking and super-resolution dSTORM microscopy. We show that LRRTM2 operates an important shift in mobility after synaptogenesis in hippocampal neurons. Knock-down of LRRTM2 during synapse formation reduced excitatory synapse density in mature neurons. Deletion of LRRTM2 C-terminal domain abolished the compartmentalization of LRRTM2 in dendrites and disrupted its synaptic enrichment. Furtheremore, we show that LRRTM2 diffusion is increased in the absence of its intracellular domain, and that the protein is more dispersed at synapses. Surprisingly, LRRTM2 confinement at synapses was strongly dependent on a YxxC motif in the C-terminal domain, but was independent of the PDZ-like binding motif ECEV. Finally, the nanoscale organization of LRRTM2 at excitatory synapses depended on its C-terminal domain, with involvement of both the PDZ-binding and YxxC motifs. Altogether, these results demonstrate that LRRTM2 trafficking and enrichment at excitatory synapses are dependent on its intracellular domain.


Assuntos
Proteínas do Tecido Nervoso , Moléculas de Adesão de Célula Nervosa , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Sinapses
4.
J Vis Exp ; (168)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33720119

RESUMO

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.


Assuntos
Neoplasias Encefálicas/patologia , Comunicação Celular , Movimento Celular , Técnicas de Cocultura/métodos , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Neurônios/patologia , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Rastreamento de Células , Glioma/patologia , Humanos , Processamento de Imagem Assistida por Computador , Laminina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia
5.
Adv Healthc Mater ; 9(18): e2000519, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32743980

RESUMO

Physiologically relevant cell-based models require engineered microenvironments which recapitulate the topographical, biochemical, and mechanical properties encountered in vivo. In this context, hydrogels are the materials of choice. Here a light-based toolbox is able to craft such microniches out of common place materials. Extensive use of benzophenone photoinitiators and their interaction with oxygen achieves this. First, the oxygen inhibition of radicals is harnessed to photoprint hydrogel topographies. Then the chemical properties of benzophenone are exploited to crosslink and functionalize native hydrogels lacking photosensitive moieties. At last, photoscission is introduced: an oxygen-driven, benzophenone-enabled reaction that photoliquefies Matrigel and other common gels. Using these tools, soft hydrogel templates are tailored for cells to grow or self-organize into standardized structures. The described workflow emerges as an effective microniche manufacturing toolset for 3D cell culture.


Assuntos
Técnicas de Cultura de Células , Hidrogéis
6.
J Cell Sci ; 133(16)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32694167

RESUMO

Cell guidance by anchored molecules, or haptotaxis, is crucial in development, immunology and cancer. Adhesive haptotaxis, or guidance by adhesion molecules, is well established for mesenchymal cells such as fibroblasts, whereas its existence remains unreported for amoeboid cells that require less or no adhesion in order to migrate. We show that, in vitro, amoeboid human T lymphocytes develop adhesive haptotaxis mediated by densities of integrin ligands expressed by high endothelial venules. Moreover, lymphocytes orient towards increasing adhesion with VLA-4 integrins (also known as integrin α4ß1), like all mesenchymal cells, but towards decreasing adhesion with LFA-1 integrins (also known as integrin αLß4), which has not previously been observed. This counterintuitive 'reverse haptotaxis' cannot be explained by existing mechanisms of mesenchymal haptotaxis involving either competitive anchoring of cell edges under tension or differential integrin-activated growth of lamellipodia, because they both favor orientation towards increasing adhesion. The mechanisms and functions of amoeboid adhesive haptotaxis remain unclear; however, multidirectional integrin-mediated haptotaxis might operate around transmigration ports on endothelia, stromal cells in lymph nodes, and inflamed tissue where integrin ligands are spatially modulated.


Assuntos
Integrinas , Antígeno-1 Associado à Função Linfocitária , Adesivos , Adesão Celular , Quimiotaxia , Humanos , Linfócitos , Molécula 1 de Adesão de Célula Vascular
7.
Elife ; 92020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31909712

RESUMO

Dynamic mechanical interactions between adhesion complexes and the cytoskeleton are essential for axon outgrowth and guidance. Whether planar cell polarity (PCP) proteins, which regulate cytoskeleton dynamics and appear necessary for some axon guidance, also mediate interactions with membrane adhesion is still unclear. Here we show that Vangl2 controls growth cone velocity by regulating the internal retrograde actin flow in an N-cadherin-dependent fashion. Single molecule tracking experiments show that the loss of Vangl2 decreased fast-diffusing N-cadherin membrane molecules and increased confined N-cadherin trajectories. Using optically manipulated N-cadherin-coated microspheres, we correlated this behavior to a stronger mechanical coupling of N-cadherin with the actin cytoskeleton. Lastly, we show that the spatial distribution of Vangl2 within the growth cone is selectively affected by an N-cadherin-coated substrate. Altogether, our data show that Vangl2 acts as a negative regulator of axonal outgrowth by regulating the strength of the molecular clutch between N-cadherin and the actin cytoskeleton.


Assuntos
Actinas/metabolismo , Caderinas/metabolismo , Proteínas do Tecido Nervoso/genética , Crescimento Neuronal/genética , Citoesqueleto de Actina/metabolismo , Animais , Cones de Crescimento/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo
8.
Sci Rep ; 10(1): 647, 2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959776

RESUMO

Accumulating evidence suggests AKT1 and DRD2-AKT-GSK3 signaling involvement in schizophrenia. AKT1 activity is also required for lithium, a GSK3 inhibitor, to modulate mood-related behaviors. Notably, GSK3 inhibitor significantly alleviates behavioral deficits in Akt1-/- female mice, whereas typical/atypical antipsychotics have no effect. In agreement with adjunctive therapy with lithium in treating schizophrenia, our data mining indicated that the average utilization rates of lithium in the Taiwan National Health Insurance Research Database from 2002 to 2013 are 10.9% and 6.63% in inpatients and outpatients with schizophrenia, respectively. Given that lithium is commonly used in clinical practice, it is of great interest to evaluate the effect of lithium on alleviating Akt1-related deficits. Taking advantage of Akt1+/- mice to mimic genetic deficiency in patients, behavioral impairments were replicated in female Akt1+/- mice but were alleviated by subchronic lithium treatment for 13 days. Lithium also effectively alleviated the observed reduction in phosphorylated GSK3α/ß expression in the brains of Akt1+/- mice. Furthermore, inhibition of Akt expression using an Akt1/2 inhibitor significantly reduced neurite length in P19 cells and primary hippocampal cell cultures, which was also ameliorated by lithium. Collectively, our findings implied the therapeutic potential of lithium and the importance of the AKT1-GSK3 signaling pathway.


Assuntos
Bases de Dados Factuais , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Seguro Saúde , Compostos de Lítio/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Esquizofrenia/tratamento farmacológico , Esquizofrenia/genética , Transdução de Sinais , Adolescente , Adulto , Idoso , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Compostos de Lítio/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Taiwan , Adulto Jovem
9.
J Psychopharmacol ; 33(10): 1288-1302, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31294644

RESUMO

BACKGROUND: Compelling animal and clinical studies support the N-methyl-D-aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia and suggest promising pharmacological agents to ameliorate negative and cognitive symptoms of schizophrenia, including sarcosine, a glycine transporter-1 inhibitor. AIMS AND METHODS: It is imperative to evaluate the therapeutic potential of sarcosine in animal models, which provide indispensable tools for testing drug effects in detail and elucidating the underlying mechanisms. In this study, a series of seven experiments was conducted to investigate the effect of sarcosine in ameliorating behavioral deficits and the underlying mechanism in pharmacological (i.e., MK-801-induced) and genetic (i.e., serine racemase-null mutant (SR-/-) mice) NMDAR hypofunction models. RESULTS: In Experiment 1, the acute administration of 500/1000 mg/kg sarcosine (i.p.) had no adverse effects on motor function and serum biochemical responses. In Experiments 2-4, sarcosine significantly alleviated MK-801-induced (0.2 mg/kg) brain abnormalities and behavioral deficits in MK-801-induced and SR-/- mouse models. In Experiment 5, the injection of sarcosine enhanced CSF levels of glycine and serine in rat brain. In Experiments 6-7, we show for the first time that sarcosine facilitated NMDAR-mediated hippocampal field excitatory postsynaptic potentials and influenced the movement of surface NMDARs at extrasynaptic sites. CONCLUSIONS: Sarcosine effectively regulated the surface trafficking of NMDARs, NMDAR-evoked electrophysiological activity, brain glycine levels and MK-801-induced abnormalities in the brain, which contributed to the amelioration of behavioral deficits in mouse models of NMDAR hypofunction.


Assuntos
Sintomas Comportamentais/tratamento farmacológico , Encefalopatias/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Glicina/agonistas , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Sarcosina/farmacologia , Esquizofrenia/tratamento farmacológico , Animais , Sintomas Comportamentais/induzido quimicamente , Encefalopatias/induzido quimicamente , Modelos Animais de Doenças , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Racemases e Epimerases/genética , Sarcosina/administração & dosagem , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Esquizofrenia/fisiopatologia
10.
Biophys J ; 112(1): 133-142, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-28076804

RESUMO

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Microscopia de Fluorescência , Fosfoproteínas/metabolismo , Linhagem Celular Tumoral , Difusão , Humanos , Fotodegradação , Fatores de Transcrição , Proteínas de Sinalização YAP
11.
J Cell Sci ; 130(1): 51-61, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27856508

RESUMO

Biomimetic materials have long been the (he)art of bioengineering. They usually aim at mimicking in vivo conditions to allow in vitro culture, differentiation and expansion of cells. The past decade has witnessed a considerable amount of progress in soft lithography, bio-inspired micro-fabrication and biochemistry, allowing the design of sophisticated and physiologically relevant micro- and nano-environments. These systems now provide an exquisite toolbox with which we can control a large set of physicochemical environmental parameters that determine cell behavior. Bio-functionalized surfaces have evolved from simple protein-coated solid surfaces or cellular extracts into nano-textured 3D surfaces with controlled rheological and topographical properties. The mechanobiological molecular processes by which cells interact and sense their environment can now be unambiguously understood down to the single-molecule level. This Commentary highlights recent successful examples where bio-functionalized substrates have contributed in raising and answering new questions in the area of extracellular matrix sensing by cells, cell-cell adhesion and cell migration. The use, the availability, the impact and the challenges of such approaches in the field of biology are discussed.


Assuntos
Materiais Biomiméticos/farmacologia , Sinais (Psicologia) , Meio Ambiente , Animais , Comunicação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos
12.
Adv Mater ; 28(10): 2024-9, 2016 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-26689426

RESUMO

Light-induced molecular adsorption of proteins (LIMAP) allows for quantitative sub-micrometer-resolution printing of multiple biomolecules. Surface-bound gradients are patterned within minutes over an entire glass cover-slip. LIMAP is used to perform selective immuno-assays, to dynamically control the adhesion of individual cells, and to achieve hierarchical co-cultures instrumental for tissue engineering.


Assuntos
Luz , Proteínas/química , Adsorção , Animais , Adesão Celular , Linhagem Celular , Fibronectinas/química , Fibronectinas/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Microscopia de Força Atômica , Microscopia de Fluorescência , Impressão Molecular , Proteínas/metabolismo , Propriedades de Superfície , Engenharia Tecidual
13.
Nat Methods ; 12(7): 641-4, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25961414

RESUMO

Single-objective selective-plane illumination microscopy (soSPIM) is achieved with micromirrored cavities combined with a laser beam-steering unit installed on a standard inverted microscope. The illumination and detection are done through the same objective. soSPIM can be used with standard sample preparations and features high background rejection and efficient photon collection, allowing for 3D single-molecule-based super-resolution imaging of whole cells or cell aggregates. Using larger mirrors enabled us to broaden the capabilities of our system to image Drosophila embryos.


Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Drosophila/embriologia
14.
Neural Dev ; 10: 5, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25888985

RESUMO

BACKGROUND: Axon pathfinding is controlled by guidance cues that elicit specific attractive or repulsive responses in growth cones. It has now become clear that some cues such as Netrin-1 can trigger either attraction or repulsion in a context-dependent manner. In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1. This finding raised the intriguing possibility that Netrin-1 and Slit1, two essential guidance cues, may act more generally in an unexpected combinatorial manner to orient specific axonal populations. To address this major issue, we have used an innovative microfluidic device compatible not only with dissociated neuronal cultures but also with explant cultures to systematically and quantitatively characterize the combinatorial activity of Slit1 and Netrin-1 on rostral thalamic axons as well as on hippocampal neurons. RESULTS: We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1. On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate. CONCLUSIONS: Our study reveals that at subthreshold repulsive levels, Slit1 acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring.


Assuntos
Axônios/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Proteínas Supressoras de Tumor/farmacologia , Animais , Axônios/classificação , Axônios/fisiologia , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Hipocampo/citologia , Humanos , Laminina/farmacologia , Camundongos , Técnicas Analíticas Microfluídicas , Proteínas do Tecido Nervoso/administração & dosagem , Netrina-1 , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Tálamo/citologia
15.
J Neurosci ; 33(45): 17647-55, 2013 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-24198357

RESUMO

The precise wiring of the nervous system relies on processes by which axons navigate in a complex environment and are guided by a concerted action of attractive and repulsive factors to reach their target. Investigating these guidance processes depends critically on our ability to control in space and time the microenvironment of neurons. The implementation of microfabrication techniques in cell biology now enables a precise control of the extracellular physical and chemical environment of cultured cells. However, microtechnology is only beginning to be applied in the field of axon guidance due to specific requirements of neuronal cultures. Here we review microdevices specifically designed to study axonal guidance and compare them with the conventional assays used to probe gradient sensing in cell biology. We also discuss how innovative microdevice-based approaches will enable the investigation of important systems-level questions on the gradient sensing properties of nerve cells, such as the sensitivity and robustness in the detection of directional signals or the combinatorial response to multiple cues.


Assuntos
Axônios/fisiologia , Cones de Crescimento/fisiologia , Neurônios/fisiologia , Neurociências/métodos , Animais , Movimento Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Neurônios/citologia , Transdução de Sinais/fisiologia
16.
Biophys J ; 103(8): 1648-56, 2012 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-23083707

RESUMO

Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. To ensure a sensitive and robust response to directional signals in complex and dynamic chemical landscapes, GCs are presumably able to amplify and filter external information. How these processing tasks are performed remains however poorly known. Here, we probe the signal-processing capabilities of single GCs during γ-Aminobutyric acid (GABA) directional sensing with a shear-free microfluidic assay that enables systematic measurements of the GC output response to variable input gradients. By measuring at the single molecule level the polarization of GABA(A) chemoreceptors at the GC membrane, as a function of the external GABA gradient, we find that GCs act as i), signal amplifiers over a narrow range of concentrations, and ii), low-pass temporal filters with a cutoff frequency independent of stimuli conditions. With computational modeling, we determine that these systems-level properties arise at a molecular level from the saturable occupancy response and the lateral dynamics of GABA(A) receptors.


Assuntos
Cones de Crescimento/fisiologia , Técnicas Analíticas Microfluídicas , Animais , Cones de Crescimento/metabolismo , Ratos , Receptores de GABA-A/metabolismo , Xenopus , Ácido gama-Aminobutírico/metabolismo
17.
Lab Chip ; 12(17): 3028-31, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22855124

RESUMO

We introduce a simple and effective method to tailor the wetting and adhesion properties of thiolene-based microfluidic devices. This one-step lithographic scheme combines most of the advantages offered by the current methods employed to pattern microchannels: (i) the channel walls can be modified in situ or ex situ, (ii) their wettability can be varied in a continuous manner, (iii) heterogeneous patterning can be easily accomplished, with contact-angle contrasts extending from 0 to 90° for pure water, (iv) the surface modification has proven to be highly stable upon aging and heating. We first characterize the wetting properties of the modified surfaces. We then provide the details of two complementary methods to achieve surface patterning. Finally, we demonstrate the two methods with three examples of applications: the capillary guiding of fluids, the production of double emulsions, and the culture of cells on adhesive micropatterns.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Animais , Carbocianinas/química , Adesão Celular , Células Cultivadas , Fibronectinas/química , Fibronectinas/metabolismo , Camundongos , Microscopia de Fluorescência , Porosidade , Propriedades de Superfície , Molhabilidade
18.
Proc Natl Acad Sci U S A ; 109(26): E1679-87, 2012 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-22689950

RESUMO

The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices--especially in optics--have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells, and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher-dimensional signals, which typically exhibits extreme redundancy. Altogether, our results emphasize the interest of CS schemes for acquisition at a significantly reduced rate and point to some remaining challenges for CS fluorescence microscopy.


Assuntos
Microscopia Confocal/métodos , Animais , Células COS , Chlorocebus aethiops , Liliaceae
19.
Lab Chip ; 12(7): 1340-6, 2012 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-22344388

RESUMO

In this paper we first introduce a novel fabrication process, which allows for easy integration of thin track-etched nanoporous membranes, within 2D or 3D microchannel networks. In these networks, soluble chemical compounds can diffuse out of the channels through well-defined and spatially organized microfabricated porous openings. Interestingly, multiple micron-scale porous areas can be integrated in the same device and each of these areas can be connected to a different microfluidic channel and reservoir. We then present and characterize several membrane-based microdevices and their use for the generation of stable diffusible concentration gradients and complex dynamic chemical landscapes under shear free conditions. We also demonstrate how a simple flow-focusing geometry can be used to generate "on-demand" concentration profiles. In turn, these devices should provide an ideal experimental framework for high throughput cell-based assays: long term high-resolution video microscopy experiments can be performed, under multiple spatially and temporally controlled chemical conditions, with simple protocols and in a cell-friendly environment.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Difusão , Dimetilpolisiloxanos/química , Fluoresceína-5-Isotiocianato/química , Membranas Artificiais , Nanoporos , Propriedades de Superfície
20.
Chem Commun (Camb) ; 47(32): 9125-7, 2011 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-21738936

RESUMO

Photoinitiated polymerisation is efficiently and rapidly carried out to immobilise ultrathin electrochemiluminescent redox hydrogel films. Microscale patterns are fabricated on an electrode surface by a simple photolithographic procedure and revealed by ECL imaging.


Assuntos
Metilgalactosídeos/química , Técnicas Eletroquímicas , Luminescência , Oxirredução , Processos Fotoquímicos , Polimerização , Propriedades de Superfície
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