Nous tenons à remercier Microsystems Laboratories technologie au MIT pour nous permettre à une partie de film de cette vidéo dans la salle blanche.

Préparation 1. Créer un masque <blockquote> Utiliser un logiciel de CAO, la conception du canal pour l&#39;impression haute résolution sur un transparent. Ce sera le &quot;masque&quot;. </blockquote> Dans la salle blanche: 2. Nettoyez et faites cuire la galette Tout d&#39;abord, la plaquette gicler avec de l&#39;acétone, puis rapidement avec du méthanol, puis à l&#39;isopropanol. Enfin, la galette sèche à l&#39;aide d&#39;azote. Cuire la galette dans le four (130 ° C) pendant 5 min. 3. Revêtement la plaquette Placer la galette au centre de la machine spin-coating. Versez résine photosensible (SU-8) de la bouteille sur le wafer. Laissez le flux SU-8 et de détente pour ~ 10 s. Allumez le spin-coucheuse et rampe jusqu&#39;à sa vitesse de 0 à 500 rpm pendant 5 s; garder à 500 rpm pendant 10 s; rampe jusqu&#39;à la vitesse finale de plus de 10 s et de maintenir à la vitesse finale de 30 s. La vitesse finale dépend de l&#39;épaisseur du revêtement ciblées et le SU-8 utilisé. Les détails peuvent être trouvés au <a href="http://www.microchem.com/">http://www.microchem.com/</a> 4. Soft-Cuire Après le revêtement de la galette, faire cuire d&#39;abord à 65 ° C, puis à 95 ° C. Le temps de cuisson varie en fonction de l&#39;épaisseur et le type de cible photosensible utilisé. Ensuite, laissez la galette reposer à température ambiante pendant au moins 5 min. 5. Exposition Placez le masque sur le dessus de la plaquette et la plaquette d&#39;exposer à la lumière UV pendant la durée recommandée de la SU-8 manuel. 6. Post-exposition de pâtisseries Cuire la galette à 65 ° C puis 95 ° C suivant les instructions de SU-8 manuel. 7. Développer la plaquette pour obtenir le &quot;maître&quot; (moule) Préparer un bécher rempli avec le développeur de méthyle (PMMA). Plonger la plaquette dans le bécher tout en oscillant très doucement le bécher jusqu&#39;à ce que la partie non exposée de la résine est lavé. Dans notre laboratoire: 8. Préparer PDMS et la verser sur la plaquette Mélanger le PDMS avec son durcisseur de 10:1 dans une tasse. Incorporer et mélanger de façon homogène c&#39;est: cela va générer beaucoup de bulles et de rendre le mélange air opaque. Verser le mélange sur le «maître». 9. De-bulle dans la chambre à vide Pour enlever les bulles, placé le mélange maître et PDMS qui est le couvrant dans une chambre à vide jusqu&#39;à ce que toutes les bulles ont disparu. 10. Cuisson au four Cuire au four pendant au moins 12 heures dans une étuve à 65 ° C pour durcir le PDMS. 11. Percez des trous Décollez le PDMS du maître et de trous de perforation pour les entrées et sorties des chaînes. En salle blanche (non montré) 12. Plasma de collage Les canaux sont collés sur une lame de verre après le traitement à la fois la couche de PDMS et la lame de verre avec un plasma d&#39;oxygène pendant 1 min. Expériences: Exp # 1: Étude de la réponse chimiotactique des microbes marins à micro-échelle couches nutriments 1) Mise en place de l&#39;expérience <ol><li> Ajouter les organismes et les substrats aux seringues en verre </li><li> Placez canal microfluidique sur platine du microscope et de joindre au tube entrées appropriées et des débouchés </li><li> Relier les tubes à perdre réservoir. Assurez-vous que le tube est entièrement immergée dans le liquide dans le réservoir de déchets pour éviter les oscillations de pression </li><li> Seringues Placer sur une pompe à seringue et raccorder aux vannes et tuyaux </li><li> Mettre en place microscope: les conditions d&#39;éclairage, grossissement, etc </li><li> Focus sur la position appropriée dans le canal </li><li> De la bulle plus grande chaîne à l&#39;aide seringue remplie d&#39;eau de mer artificielle </li><li> Réglez le débit approprié sur une pompe à seringue. Dans ce cas, 2 ml / min, ce qui correspond à une vitesse moyenne d&#39;écoulement de -1 220 um s dans le canal </li></ol> 2) Exécution de l&#39;expérience <ol><li> Démarrer la pompe seringue pour établir un gradient de nutriments dans le canal </li><li> Une fois de flux s&#39;est stabilisé et une bande de nutriments a développé, arrêter le flux de la pompe seringue et commencer à enregistrer le temps de ce point </li><li> Bande de nutriments commence à diffuser latéralement </li><li> A intervalles de temps réguliers, utiliser un logiciel d&#39;analyse d&#39;image pour enregistrer des séquences d&#39;images pour créer des «films» </li><li> Discriminer les organismes de natation en prenant le temps de différence d&#39;images entre deux images suivantes, de sorte que seuls les objets en mouvement sont maintenant visualisées, ce qui nous permet de différencier les cellules mobiles du non-déplacement des particules et de bruit de fond </li><li> Prenez les films pour déterminer les positions des cellules dans le canal en référence à la position du patch nutriments </li><li> Enregistrez des vidéos à intervalles réguliers pendant 10-20 min à analyser les positions et les habitudes de nage des organismes </li><li> En utilisant le logiciel d&#39;analyse d&#39;image à superposer les positions des organismes dans différents cadres (en attribuant à chaque pixel de l&#39;intensité lumineuse maximale enregistrée dans ce pixel sur la durée du film), nous pouvons obtenir des informations de trajectoire pour les cellules de la natation </li></ol> Exp # 2: Étudier les effets de cisaillement sur des bactéries marines nageant dans un vortexZ <ol><li> En utilisant la géométrie canal différent, nous pouvons observer le comportement des bactéries nageant dans un vortex à différents taux de cisaillement </li></ol>

<ol>
	<li>Azam, F., Malfatti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+structuring+of+marine+ecosystems.">Microbial structuring of marine ecosystems.</a> Nature Reviews Microbiology. 5, 782-791 (2007).</li><li>Blackburn, N., Azam, F., Hagstrom, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+explicit+simulations+of+a+microbial+food+web.+.">Spatially explicit simulations of a microbial food web. .</a> Limnology and Oceanography. 42, 613-622 (1997).</li><li>Blackburn, N., Fenchel, T., Mitchell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+nutrient+patches+in+plankton+habitats+shown+by+chemotactic+bacteria.">Microscale nutrient patches in plankton habitats shown by chemotactic bacteria.</a> Science. 282, 2254-2256 (1998).</li><li>Keymer, J. E., Galajda, P., Muldoon, C., Park, S., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+metapopulations+in+nanofabricated+landscapes.">Bacterial metapopulations in nanofabricated landscapes.</a> Proceedings of the National Academy of Science. 103, 17290-17295 (2006).</li><li>Mao, H., Cremer, P. S., Manson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive,+versatile+microfluidic+assay+for+bacterial+chemotaxis.">A sensitive, versatile microfluidic assay for bacterial chemotaxis.</a> Proceedings of the National Academy of Science. 100, 5449-5454 (2003).</li><li>Marcos, R., Stocker, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microorganisms+in+vortices:+a+microfluidic+setup.">Microorganisms in vortices: a microfluidic setup.</a> Limnology and Oceanography: Methods. 4, 392-398 (2006).</li><li>Park, S., Wolanin, P. M., Yuzbahyan, E. A., Lin, H., Darnton, N. C., Stock, J. B., Silberzan, P., Austin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+topology+on+bacterial+social+interaction.">Influence of topology on bacterial social interaction.</a> Proceedings of the National Academy of Sciences. 100, 13910-13915 (2003).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annual Review of Biomedical Engineering. 3, 335-373 (2001).</li></ol>

Une compréhension de comment les microbes marins interagissent avec leur composition chimique locale et l&#39;environnement physique est indispensable pour une perception plus complète et précise du rôle des micro-organismes planctoniques dans les océans des nutriments et de carbone de cycles (Azam et Malfatti 2007). Toutefois, en raison de la petite échelle (mm &lt;) sur lequel de nombreuses interactions microbiennes importantes ont lieu, les limites techniques ont empêché un examen détaillé du comportement microbien dans le hét...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PDMS, Sylgard 184</td>
<td>Silicone Elastomer Kit</td>
<td>Dow Corning, Midland, MI, USA</td>
<td>&nbsp;</td>
<td>http://www.ellsworth.com/sylgard.html</td>
</tr>
<tr>
<td>SU8-2100</td>
<td>Photoresist</td>
<td>MicroChem, Newton, MA, USA</td>
<td>&nbsp;</td>
<td>www.microchem.com
</td>
</tr>
<tr>
<td>Nikon Eclipse TE2000-E inverted microscope</td>
<td>Microscope</td>
<td>Nikon, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEEK tubing (0.762 mm ID, 1.59 mm OD)</td>
<td>Tool</td>
<td>Upchurch Scientific, Oak Harbor, WA, USA</td>
<td>&nbsp;</td>
<td>www.upchurch.com</td>
</tr>
<tr>
<td>Syringes (Luer-Lok Tip)</td>
<td>Tool</td>
<td>BD, Franklin Lakes, NJ, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fitting Part P-704-01</td>
<td>Tool</td>
<td>Upchurch Scientific, Oak Harbor, WA, USA</td>
<td>&nbsp;</td>
<td>To connect tubing to Luer-Lok Tip Syringes</td>
</tr>
<tr>
<td>Syringe Pump (PHD 2000 Programmable)</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CCD Camera (PCO 1600)</td>
<td>Equipment</td>
<td>Cooke, Romulus, MI, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

chemotactic response of marine micro-organisms to micro-scale nutrient layers

La mesure dans laquelle les microbes planctoniques peuvent exploiter des parcelles de ressources micro-aura des implications considérables pour trophodynamique océaniques et flux biogéochimiques. Cependant, pour profiter de correctifs de nutriments dans l&#39;océan, les microbes de natation doit surmonter l&#39;influence de forces physiques, y compris la diffusion moléculaire et turbulente de cisaillement, ce qui limite la disponibilité de correctifs et de la capacité des bactéries à les localiser. Jusqu&#39;à récemment, les limites méthodologiques ont empêché les examens directs de comportement microbien dans des habitats fragmentaires et réaliste des conditions d&#39;écoulement à petite échelle. Ainsi, beaucoup de nos connaissances actuelles concernant le comportement microbien dans l&#39;océan a été obtenue à partir des prédictions théoriques. Pour obtenir de nouvelles informations sur le comportement alimentaire microbien dans l&#39;océan, nous avons appliqué les techniques de fabrication souples lithographiques pour développer deux dispositifs microfluidiques, que nous avons utilisés pour créer des (i) micro-nutriments patchs avec des dimensions et des caractéristiques de diffusion pertinents pour les processus océaniques et (ii) micro- tourbillons, avec des taux de cisaillement correspondant à ceux attendus dans l&#39;océan. Ces dispositifs microfluidiques ont permis un premier examen direct de la natation et le comportement microbien dans un paysage chimiotactiques hétérogènes et dynamiques. L&#39;utilisation combinée de la microscopie à épifluorescence et contraste de phase permet d&#39;examens directs des dimensions physiques et caractéristiques de diffusion de correctifs en éléments nutritifs, tout en observant la réponse agrégative niveau de la population, en plus du comportement de nage des microbes individuels. Ces expériences ont révélé que certaines espèces de phytoplancton, bactéries hétérotrophes et les protistes phagotrophic sont aptes à repérer et exploiter les ressources de diffusion des correctifs microscopique dans des délais très courts. Nous avons également montré que jusqu&#39;à modérée des taux de cisaillement, les bactéries marines sont capables de lutter contre le flux et nager à travers leur environnement à leur propre gré. Cependant, au-delà d&#39;un niveau de cisaillement seuil élevé, les bactéries sont alignés dans le flux de cisaillement et sont moins capables de nager sans perturbation de l&#39;écoulement. Microfluidique représente une approche novatrice et peu coûteuse pour l&#39;étude de l&#39;écologie microbienne aquatique, et en raison de son aptitude à créer des champs d&#39;écoulement précision réaliste et gradients substrat à l&#39;échelle microscopique, est idéalement applicable aux examens du comportement microbien à la plus petite des échelles d&#39;interaction. Nous suggérons donc que la microfluidique représente un outil précieux pour l&#39;obtention d&#39;une meilleure compréhension de l&#39;écologie des microorganismes dans l&#39;océan.

Watch this Scientific Journal Video about Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers at JoVE.com

Meiosis  | Biology Video Collection

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

La fabrication des canaux microfluidiques et de leur mise en œuvre dans des expériences pour étudier le comportement chimiotactique butinage des microbes marins dans un paysage parcellaire des éléments nutritifs et le comportement de nage des bactéries dans les flux de cisaillement sont décrits.

Réponse chimiotactique des micro-organismes marins à l&#39;échelle micro nutriments des couches

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

chemotactic-response-marine-micro-organisms-to-micro-scale-nutrient

Research

JoVE Journal

Biology

13.2K vues.  MIT - Massachusetts Institute of Technology. La fabrication des canaux microfluidiques et de leur mise en œuvre dans des expériences pour étudier le comportement chimiotactique butinage des microbes marins dans un paysage parcellaire des éléments nutritifs et le comportement de nage des bactéries dans les flux de cisaillement sont décrits.

Vidéo: Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth.  Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Couches de Symbiose - Visualisation de la communauté microbienne Termite Intestin postérieur

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different ...

Recherche

Biologie

Micro-scale Engineering for Cell Biology

Micro-échelle d&#39;ingénierie de biologie cellulaire

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

L&#39;imagerie in vivo de Deep couches corticales utilisant un microprisme

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

En utilisant la réponse optocinétique pour étudier la fonction visuelle du poisson zèbre

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantification des γH2AX Foci en réponse aux radiations ionisantes

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Mesure de temps de survie dans<em&gt; Brachionus</em&gt; Rotifères: Synchronisation des conditions maternelles

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hydroponique: un système polyvalent pour étudier et répartition des plantes Réponses des éléments nutritifs à la disponibilité des éléments nutritifs et l&#39;exposition aux éléments toxiques

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Caractérisation de calcification Events Utilisation en direct optiques et Electron Microscopy Techniques dans un tubeworm Marine

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

La détection de G Protein-Coupled Receptor Internalisation activé de ligand par microscopie confocale

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Évaluation de Dictyostelium discoideum réponse à la Stimulation mécanique aiguë

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...