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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. ispartof: BIOLOGY OF THE CELL vol:113 issue:12 pages:492-506 ispartof: location:England status: published

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 (

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.

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.

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/β 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.

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 α4β1), like all mesenchymal cells, but towards decreasing adhesion with LFA-1 integrins (also known as integrin αLβ4), 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.

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

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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.

3D cell culture aims at reconciliating the simplicity of in vitro models with the human like properties encountered in vivo. Soft permeable hydrogels have emerged as user-friendly materials to grow cells in more physiological conditions. With the intent on turning these homogeneous substrates into biomimetic templates, we introduce a generic solution compatible with the most biologically relevant and often frail materials. Here we take control of the chemical environment driving generic radical reactions to craft common gels with patterned light. In a simple microreactor, we harness the well-known inhibition of radicals by oxygen to enable topographical photopolymerization. Strikingly, by sustaining an oxygen rich environment, we can also induce hydrogel photo-scission which turns out to be a powerful and generic subtractive manufacturing method. We finally introduce a flexible patterned functionalization protocol based on available photo-linkers. Using these common tools on the most popular hydrogels, we tailored soft templates where cells grow or self-organize into standardized structures. The platform we describe has the potential to set a standard in future 3D cell culture experiments.

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.

The in vitro methods to recapitulate environmental cues around cells are usually optimized to test a specific property of the environment (biochemical nature or the stiffness of the extra cellular matrix (ECM), or nanotopography) for its capability to induce defined cell behaviors (lineage commitment, migration). Approaches that combine different environmental cues in 3D to assess the biological response of cells to the spatial organization of different biophysical and biochemical cues are growingly being developed. We demonstrate how the lamination of through-hole polymeric bio-functionalized membranes can be implemented to create complex bona fide micro-niches with differential 3D environmental properties using photoactive materials. Our approach enables to create micro-niches ranging in size from single cells to cell aggregates. They are bio-functionalized in 3D simultaneously with topographical featured, protein patterns and structured ECM surrogate with 1 micrometer resolution. We demonstrate how these niches extend in 3D the ability to pattern cells. We exemplify how they can be used to standardize cells shapes in 3D and to trigger the apico-basal polarization of single epithelial cells.

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.

Printing of biomolecules on substrates has developed tremendously in the past few years. The existing methods either rely on slow serial writing processes or on parallelized photolithographic techniques where cumbersome mask alignment procedures usually impair the ability to generate multi-protein patterns. We recently developed a new technology allowing for high resolution multi protein micro-patterning. This technology named “Light-Induced Molecular Adsorption of Proteins (LIMAP)” is based on a water-soluble photo-initiator able to reverse the antifouling property of polymer brushes when exposed to UV light. We developed a wide-field pattern projection system based on a DMD coupled to a conventional microscope which permits to generate arbitrary grayscale patterns of UV light at the micron scale. Interestingly, the density of adsorbed molecules scales with the dose of UV light thus allowing the quantitative patterning of biomolecules. The very low non specific background of biomolecules outside of the UV-exposed areas allows for the sequential printing of multiple proteins without alignment procedures. Protein patterns ranging from 500 nm up to 1 mm can be performed within seconds, as well as gradients of arbitrary shapes. The range of applications of the LIMAP approach extends from the single molecule up to the multicellular scale with an exquisite control over local protein density. We show that it can be used to generate complex protein landscapes useful to study protein-protein, cell-cell and cell-matrix interactions.

Induced-charge electro-osmosis (ICEO) is demonstrated around an isolated platinum wire in a polymer microchannel filled with lowconcentration KCl, subject to a weak alternating electric field. In contrast to ac electro-osmosis at electrode arrays, which shares the same slip mechanism, ICEO has a more general frequency dependence, including steady flow in the dc limit (since the wire is not an electrode driving the field). The flow profile inside the device, measured by particle image velocimetry, confirms the predicted scaling with the square of the applied voltage, as well as the characteristic cut-off frequency. A quantitative comparison with numerical solutions of the models equations is reported for various equivalent circuit models from the literature. The standard model of linear capacitors captures the basic trends, but systematically over-predicts the velocity. A better fit to experiment can be obtained with a complex impedance, taking into account the frequency dispersion of capacitance, although the microscopic justification of this model is unclear especially in the presence of electro-osmotic flow and applied voltages well into the non-linear regime. © 2005 Elsevier B.V. All rights reserved.

Accurate determination of the mechanical response of pneumatic activated microvalves is important for the optimization of a number of microfluidic elements. In the case of multi-layers PDMS devices, pneumatic activated valves are used in combination for pumping, mixing and other functionalities. In such devices, the mechanical response of a valve depends on the PDMS toughness, the membrane thickness as well as the detail of the channel configuration and the pressure applied in both flow and control channels. We describe characterization technique of a pneumatic activated valve based on conductance measurements of electrolytes in the microfluidic channel. We also show that the well calibrated microvalves can be used for the development of more complex functionalities such as high precision dose control of chemical solutions.

The purification of nucleic acids from microbial and mammalian cells is a crucial step in many biological and medical applications1. We have developed microfluidic chips for automated nucleic acid purification from small numbers of bacterial or mammalian cells. All processes, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment. Measurable amounts of mRNA were extracted in an automated fashion from as little as a single mammalian cell and recovered from the chip. These microfluidic chips are capable of processing different samples in parallel, thereby illustrating how highly parallel microfluidic architectures can be constructed to perform integrated batch-processing functionalities for biological and medical applications.

High-density microfluidic chips contain plumbing networks with thousands of micromechanical valves and hundreds of individually addressable chambers. These fluidic devices are analogous to electronic integrated circuits fabricated using large scale integration (LSI). A component of these networks is the fluidic multiplexor, which is a combinatorial array of binary valve patterns that exponentially increases the processing power of a network by allowing complex fluid manipulations with a minimal number of inputs. These integrated microfluidic networks can be used to construct a variety of highly complex microfluidic devices, for example the microfluidic analog of a comparator array, and a microfluidic memory storage device resembling electronic random access memories.

Two techniques for the fabrication of novel microfluidic devices for electrokinetic fluid pumping are presented. Both consist of forming a micro-channel and reservoirs on a transparent cover sheet and patterning an array of interdigitated asymmetric micro-electrodes on a flat substrate. The two techniques differ from each other in their building materials as well as the pattern replication technique used for the cover sheet fabrication. In the first approach, we used a glass substrate for the electrode fabrication and casting of an elastomer for the cover sheet. In the second approach, both cover sheet and substrate are obtained by imprinting plastic pellets on a pre-patterned or flat mold. In assembled devices, pumping has been demonstrated and characterized by optical microscopy. The observed dependence of the pumping velocity on applied voltage and frequency are in line with theoretical predictions.

We demonstrate that room-temperature and low-pressure nanoimprint lithography techniques can be achieved by using Hybrane HS2550, a semi-crystalline hyperbranched resist polymer with a glass transition temperature below and a melting point above room temperature. Nanoimprint lithography at room temperature is possible with sub-100 nm resolution, as 75 nm line-and-spacing gratings were successfully fabricated with a tri-layer process and a metal lift-off. The melt viscosity of Hybrane HS2550 decreases drastically with temperature allowing nanoimprint experiments at low pressures, while maintaining imprint temperatures that are much lower than commonly required in nanoimprint technology.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13064-015-0036-8) contains supplementary material, which is available to authorized users.

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.

Difficulties in culturing cells inside microchannels is a major obstacle for the wide use of microfluidic technology in cell biology. Here, we present a simple and versatile method to interface closed microchannels with cellular and multicellular systems. Our approach, based on microfluidic stickers which can adhere to wet glass coverslips, eliminates the need to adapt cell culture conditions to microchannels and greatly facilitates the methods required to position cells into microcircuits. We demonstrate the simplicity and efficiency of the method with HeLa cells, primary cultured neurons and Drosophila tissues.

Single particle tracking (SPT) techniques such as sptPALM, uPAINT, and quantum dot tracking have given unprecedented insight into molecular dynamics in living cells. They allow monitoring the behavior of individual proteins in the plasma membrane as well as their molecular interaction with scaffold proteins at millisecond temporal resolution and high spatial resolution (

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.

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.

In fluorescence microscopy, one can distinguish two kinds of imaging approaches, wide field and raster scan microscopy, differing by their excitation and detection scheme. In both imaging modalities the acquisition is independent of the information content of the image. Rather, the number of acquisitions N, is imposed by the Nyquist-Shannon theorem. However, in practice, many biological images are compressible (or, equivalently here, sparse), meaning that they depend on a number of degrees of freedom K that is smaller that their size N. Recently, the mathematical theory of compressed sensing (CS) has shown how the sensing modality could take advantage of the image sparsity to reconstruct images with no loss of information while largely reducing the number M of acquisition. Here we present a novel fluorescence microscope designed along the principles of CS. It uses a spatial light modulator (DMD) to create structured wide field excitation patterns and a sensitive point detector to measure the emitted fluorescence. On sparse fluorescent samples, we could achieve compression ratio N/M of up to 64, meaning that an image can be reconstructed with a number of measurements of only 1.5 % of its pixel number. Furthemore, we extend our CS acquisition scheme to an hyperspectral imaging system.

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 out to some remaining challenges for CS fluorescence microscopy., Comment: Submitted to Proceedings of the National Academy of Sciences of the United States of America

International audience; 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 ?-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. Cop. 2012 Biophysical Society.

3 pages; International audience; 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.

5 pages; International audience; Immobilization and electrical wiring of enzymes is of particular importance for the elaboration of efficient biosensors and can be cumbersome. Here, we report a fast and easy protocol for enzyme immobilization, and as a proof of concept, we applied it to the immobilization of bilirubin oxidase, a labile enzyme. In the first step, bilirubin oxidase is mixed with a redox hydrogel "wiring" the enzyme reaction centers to electrodes. Then, this adduct is covered by an outer layer of PEGDA made by photoinitiated polymerization of poly(ethylene-glycol) diacrylate (PEGDA) and a photoclivable precursor, DAROCUR. This two-step protocol is 18 times faster than the current state-of-the-art protocol and leads to currents 25% higher. In addition, the outer layer of PEGDA acts as a protective layer increasing the lifetime of the electrode by 100% when operating continuously for 2000 s and by 60% when kept in dry state for 24 h. This new protocol is particularly appropriate for labile enzymes that quickly denaturate. In addition, by tuning the ratio PEGDA/DAROCUR, it is possible to make the enzyme electrodes even more active or more stable.

Fundamental and applied biomedical research increasingly relies on the quantitative analysis of the response of individual cells to controlled stimulations. The advent of microfluidics has revolutionized the methods to tailor the chemical environment of live cells, allowing stimulations to be applied with high spatiotemporal control and using minute amounts of reagents. It has been shown that Dictyostelium Discoideum cells (D.D.) can sense cAMP gradients on scales smaller than their size (a few tens of microns) and can adapt to very rapid concentration changes on sub second timescales. Classical methods to apply chemical stimulations, which are often based on the diffusion of solutes released by micropipettes, suffer from a poor spatial and temporal resolution and cannot discriminate the different timescales involved in the gradient sensing mechanism of D.D. Here we take advantage of a recently developed microfluidic toolbox based on microfluidic stickers made out of stiff polymers to stimulate adherent D.D. cells within microchannels. Microfluidic stickers allow for periodic chemical stimulation at the subcellular scale at frequencies up to 10Hz.We will present the dependence of the gradient sensing response as a function of both the temporal and spatial frequency. We will show how the microfluidic framework used in this study overcomes several experimental limitations and thus considerably extends previous investigations on the chemotaxis of D.D. by giving a new insight on previously proposed gradient sensing models.

Sensitivity to weak directional signals is a striking feature of chemotactic systems. In eukaryotic cells, it is often attributed to spatial amplification in the detection of the gradient of guidance cues due to an asymmetric distribution of signaling molecules of the signaling pathway. By combining single-quantum dot imaging with a guidance assay, we probed the dynamics of GABAA receptors (GABAARs) in nerve growth cones. In presence of a GABA gradient, we observed a lateral redistribution of the receptors towards the GABA source. This effect was both reversible and specific of GABA signals. Its functional implication was established by calcium imaging which showed that the redistribution was accompanied by an enhanced asymmetry of the calcium response. Furthermore, single quantum-dot tracking of GABAARs revealed a “conveyor-belt” type of motion in which receptors randomly alternated between periods of free diffusion and of microtubule-dependent directed movement.We therefore propose a model in which an asymmetric activation by the signaling gradient leads to oriented growth of the MTs which, in turn, contributes to an asymmetric distribution of the receptors and amplification in gradient sensing. This simple model for the formation of polarity at the cell membrane is supported by numerical simulations that describe with minimal hypothesis the results of our experiments. These simulations also provide predictions on the dependence of the formation of polarity as a function of gradient parameters. We will finally show our current effort to place neurons in microfluidic devices, to generate controlled gradients and to characterize the growth cone as a sensing, amplifying and filtering module.

We study microfluidic systems in which mass exchanges take place between moving water droplets, formed on-chip, and an external phase (octanol). Here, no chemical reaction takes place, and the mass exchanges are driven by a contrast in chemical potential between the dispersed and continuous phases. We analyze the case where the microfluidic droplets, occupying the entire width of the channel, extract a solute-fluorescein-from the external phase (extraction) and the opposite case, where droplets reject a solute-rhodamine-into the external phase (purification). Four flow configurations are investigated, based on straight or zigzag microchannels. Additionally to the experimental work, we performed two-dimensional numerical simulations. In the experiments, we analyze the influence of different parameters on the process (channel dimensions, fluid viscosities, flow rates, drop size, droplet spacing, ...). Several regimes are singled out. In agreement with the mass transfer theory of Young et al. (Young, W.; Pumir, A.; Pomeau, Y. Phys. Fluids A 1989, 1, 462), we find that, after a short transient, the amount of matter transferred across the droplet interface grows as the square root of time and the time it takes for the transfer process to be completed decreases as Pe-2/3, where Pe is the Peclet number based on droplet velocity and radius. The numerical simulation is found in excellent consistency with the experiment. In practice, the transfer time ranges between a fraction and a few seconds, which is much faster than conventional systems.

To correlate gene expression profiles to fundamental biological processes such as cell growth, differentiation and migration, it is essential to work at the single cell level. Gene expression analysis always starts with the relatively low efficient reverse transcription (RT) of RNA into complementary DNA (cDNA), an essential step as unprocessed RNAs will not be analysed further. In this paper, we present a novel method for RT that uses microfluidics to manipulate nanolitre volumes. We compare our method to conventional protocols performed in microlitre volumes. More specifically, reverse transcription was performed either in a polydimethylsiloxane (PDMS) rotary mixer or in a tube, using a single cell amount of mouse brain RNA (10 pg), and was followed by a template-switching PCR (TS-PCR) amplification step. We demonstrate that, using the microfluidic protocol, 74% of the genes expressed in mouse brain were detected, while only 4% were found with the conventional approach. We next profiled single neuronal progenitors. Using our microfluidic approach, i.e. performing cell capture, lysis and reverse transcription on-chip followed by TS-PCR amplification in tube, a mean of 5000 genes were detected in each neuron, which corresponds to the expected number of genes expressed in a single cell. This demonstrates the outstanding sensitivity of the microfluidic method.

We present how to make and assemble micro-patterned stickers (microPS) to construct high performance plastic microfluidic devices in a few minutes. We take advantage of soft UV imprint techniques to tailor the geometry, the mechanical properties, and the surface chemistry of 2D and 3D microfluidic circuits. The resulting microfluidic stickers substantially overcome the actual performance of the very popular PDMS devices for a wide range of applications, while sharing their celebrated fast and easy processing. To highlight the intrinsic advantages of this method, three important applications are detailed: (i) we show that both aqueous and organic droplets can be produced and stored in stickers without any specific surface coating. (ii) We report on the outstanding pressure resistance of the microPS, which open the way to the transport of viscous complex fluids. (iii) Finally, a simple design strategy is proposed to generate complex flow patterns in interconnected stacks of microPS.

We have built a dedicated lab on a chip to study the performance of an integrated electrokinetic micropump, driven by a low voltage AC signal. This micropump consists of an array of interdigitated electrodes and is here integrated in a microfluidic loop. We demonstrate that this device can pump continuously and reproducibly electrolyte solutions of low to moderate ionic strength. The pumping speed reaches up to 500 [micro sign]m s(-1) in 20 [micro sign]m deep and 100 [micro sign]m wide channels with a driving signal in the 1-10 kHz range and an amplitude of only a few volts. In addition, we have observed an interesting reversal of the pumping direction at higher frequencies (50-100 kHz). Our device permits a systematic and automated exploration of the influence of the ionic strength thanks to an integrated micromixer.

Using basic physical arguments, we present a design and method for the fabrication of microfluidic valves using multilayer soft lithography. These on-off valves have extremely low actuation pressures and can be used to fabricate active functions, such as pumps and mixers in integrated microfluidic chips. We characterized the performance of the valves by measuring both the actuation pressure and flow resistance over a wide range of design parameters, and compared them to both finite element simulations and alternative valve geometries.

We have used nanoimprint-based techniques [1] to fabricate capillary electrophoresis chips with an in-channel array of nanopillars for DNA separation, in both silicon dioxide and plastic substrates.

In the present work, we have measured the messenger RNA expression of specific genes both from total RNA and cells encapsulated in droplets. The microfluidic chip introduced includes the following functionalities: RNA∕cell encapsulation, lysis, reverse transcription and real-time polymerase chain reaction. We have shown that simplex and duplex gene expression measurements can be carried out over a population of 100 purified RNA samples encapsulated simultaneously in 2 nl droplets in less than 2 h. An analysis of 100 samples containing one to three cells has shown excellent consistency with standard techniques regarding average values. The cell-to-cell distributions of the E-cadherin expression suggest fluctuations on the order of 80% in the number of transcripts, which is highly consistent with the general findings from the literature. A mathematical model has also been introduced to strengthen the interpretation of our results. The present work paves the way for the systematic acquisition of such information in biological and biomedical studies.

We introduce a simple and versatile microfluidic drop-on-demand solution that enables independent and dynamical control of both the drop size and the drop production rate. To do so, we combine a standard microfluidic T- junction and a novel active switching component that connects the microfluidic channel to the macroscopic liquid reservoirs. Firstly, we explain how to make this simple but accurate drop-on-demand device. Secondly, we carefully characterize its dynamic response and its range of operations. Finally, we show how to generate complex two-dimensional drop patterns dynamically in single or multiple synchronized drop-on-demand devices.

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.

We present a new imaging technique using surface-plasmon mediated fluorescence microscopy. It uses a similar configuration as standard prismless Total Internal Reflection Fluorescence Microscopy with an additional metallic thin film. In the case of a silver thin film we show that this technique offers many advantages: distance dependence emission filter for improved signal to noise ratio and enhanced molecular detection efficiency. This technique is of particular interest in membrane and adhesion imaging. We present real time images on live cells.