Wir möchten Microsystems Technology Laboratories am MIT für die Erlaubnis, Film Teil dieses Video in den Reinraum zu danken.

Vorbereitung 1. Erstellen Sie eine Maske <blockquote> Mit Hilfe eines CAD-Software, Design der Kanal für den hochauflösenden Druck auf Transparenz. Dies wird die &quot;Maske&quot; werden. </blockquote> Im Reinraum: 2. Saubere und backen den Wafer Zuerst spritzt der Wafer mit Aceton, dann schnell mit Methanol, dann mit Isopropanol. Zum Schluß wird der Wafer mit Stickstoff. Backen Sie die Wafer in den Ofen (130 ° C) für 5 min. 3. Die Beschichtung der Wafer Legen Sie die Wafer in der Mitte der Spin-Coating-Maschine. Gießen Photolack (SU-8) aus der Flasche auf den Wafer. Lassen Sie die SU-8 fließen und entspannen Sie sich für ~ 10 s. Schalten Sie den Spin-Coater und Rampe seine Geschwindigkeit bis 0 bis 500 Umdrehungen pro Minute mehr als 5 s; halten bei 500 rpm für 10 s; Rampe bis zur endgültigen Geschwindigkeit über 10 s und unterhalten auf Endgeschwindigkeit für 30 s. Die Endgeschwindigkeit hängt von der gezielten Schichtdicke und dem SU-8 verwendet. Die Details finden Sie unter <a href="http://www.microchem.com/">http://www.microchem.com/</a> 4. Soft-backen Nach der Beschichtung der Wafer, backen sie zunächst bei 65 ° C und dann bei 95 ° C. Die Backzeit variiert mit gezielten Dicke und Art des verwendeten Photolack. Dann lassen Sie den Wafer bei Raumtemperatur stehen lassen für mindestens 5 min. 5. Belichtung Legen Sie die Maske auf dem Wafer und setzen die Wafer mit UV-Licht für die Zeit in der SU-8 Bedienungsanleitung empfohlen. 6. Post-Exposure Bake Backen Sie die Wafer bei 65 ° C und dann 95 ° C im Anschluss an die SU-8 Beschreibung in der Bedienungsanleitung. 7. Die Entwicklung der Wafer auf die &quot;Master&quot; (Schimmel) zu erhalten Bereiten Sie ein Becherglas mit dem Entwickler (PMMA) ausgefüllt. Tauchen Sie die Wafer in den Becher, während sehr sanft schwingenden das Becherglas, bis die unbelichteten Teil der Fotolack wird weggespült. In unserem Labor: 8. Bereiten PDMS und gieße es auf den Wafer Mischen Sie die PDMS mit seinen Härter im Verhältnis 10:1 in eine Tasse. Umrühren und mischen Sie es homogen: Dies wird viele Blasen zu erzeugen und die Mischung Blick undurchsichtig. Gießen Sie die Mischung auf dem &quot;Master&quot;. 9. De-Blase in der Vakuumkammer Zum Entfernen der Blasen, legte der Meister und PDMS-Mischung, die für sie in einer Vakuumkammer, bis alle Luftblasen verschwunden sind. 10. Backen im Ofen Backen Sie für mindestens 12 Stunden in einem Ofen bei 65 ° C zum Aushärten des PDMS. 11. Lochen Ziehen Sie das PDMS von der Master-und Lochung für Ein-und Auslässe der Kanäle. Im Reinraum (nicht abgebildet) 12. Plasma Bindung Die Kanäle werden auf einem Glasträger nach der Behandlung sowohl die PDMS-Schicht und der Objektträger aus Glas mit Sauerstoff-Plasma für 1 min verbunden. Experimente: Exp # 1: Untersuchung der chemotaktischen Reaktion der marinen Mikroben zu Mikro-Maßstab Nährstoff-Schichten 1) Einrichten des Tests <ol><li> Add Organismen und Substrate Glasspritzen </li><li> Legen Sie mikrofluidischen Kanal auf Mikroskop-Bühne und legen Schläuche, geeignete Ein-und Auslässe </li><li> Die Leitungen zum Speicher verschwenden. Stellen Sie sicher, Schlauch vollständig in der Flüssigkeit in Abfallbehälter untergetaucht, um Druckschwankungen zu vermeiden </li><li> Legen Spritzen auf Spritzenpumpe und die Ventile und Schläuche verbinden </li><li> Set up Mikroskop: Lichtverhältnisse, Vergrößerung, etc. </li><li> Konzentrieren Sie sich auf entsprechende Position in den Kanal </li><li> De-bubble-Kanal mit größeren Spritze mit künstlichem Meerwasser gefüllt </li><li> Festlegen geeigneter Durchflussrate auf Spritzenpumpe. In diesem Fall 2 ml / min, was einer mittleren Strömungsgeschwindigkeit von 220 um s -1 entspricht in den Kanal </li></ol> 2) Ausführung des Experiments <ol><li> Starten Spritzenpumpe ein Nährstoff-Gradienten in den Kanal zu etablieren </li><li> Sobald flow hat sich stabilisiert und eine Bande von Nährstoffen entwickelt hat, stoppen Sie den Fluss auf die Spritzenpumpe und beginnt die Aufnahme an diesem Punkt </li><li> Nutrient Band beginnt seitlich diffuse </li><li> In regelmäßigen Zeitabständen, verwenden Bildanalyse-Software, um Sequenzen von Bildern aufnehmen zu &quot;Filme&quot; zu schaffen </li><li> Diskriminieren Schwimmen Organismen, indem sie Zeit-Differenz Bilder zwischen zwei aufeinander folgenden Frames, so dass nur bewegte Objekte sind nun sichtbar, so dass wir bewegliche Zellen aus nicht-bewegenden Teilchen und Hintergrundgeräuschen unterscheiden </li><li> Nehmen Sie Filme auf Positionen von Zellen in den Kanal mit Verweis auf die p bestimmenosition des Nährstoffs Patch </li><li> Nehmen Sie die Videos in regelmäßigen Abständen für 10-20 min zur Analyse der Positionen und Schwimmen Muster von Organismen </li><li> Mit Hilfe der Bildanalyse-Software, um die Positionen von Organismen in verschiedenen Frames (Zuordnung, die jedem Pixel die maximale Lichtintensität in diesem Pixel über die Dauer des Films aufgezeichnet) überlagern, können wir Flugbahn Informationen zum Schwimmen Zellen zu erhalten </li></ol> Exp # 2: Untersuchung der Auswirkungen der Scherung auf marinen Bakterien schwimmen in einem vortexZ <ol><li> Mit verschiedenen Kanalgeometrie, können wir beobachten das Verhalten von Bakterien schwimmen in einem Wirbel bei unterschiedlichen Schergeschwindigkeiten </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>

Ein Verständnis davon, wie marine Mikroben Interaktion mit den lokalen chemischen und physikalischen Umwelt ist für eine vollständige und genaue Wahrnehmung der Rolle der planktonischen Mikroorganismen in den Ozeanen Nährstoff-und Kohlenstoff-Zyklen (Azam und Malfatti 2007) unbedingt erforderlich. Aufgrund der kleinen Skalen (&lt;mm), über die viele wichtige mikrobielle Interaktionen stattfinden, technischen Einschränkungen haben detaillierte Untersuchungen der mikrobiellen Verhalten innerhalb der heterogenen bio-physikalisch-chemisch...

<!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

Der Grad der planktonischen Mikroben mikroskaligen Ressource Patches nutzen können, haben erhebliche Auswirkungen auf ozeanischen trophodynamics und biogeochemischen Fluss. Doch die Vorteile der Nährstoff-Patches in den Ozean, Schwimmen Mikroben müssen die Einflüsse der physischen Kräfte, einschließlich der molekularen Diffusion und turbulente Scherung, die die Verfügbarkeit von Patches und die Fähigkeit der Bakterien, um sie ausfindig zu begrenzen überwinden. Bis vor kurzem haben methodische Einschränkungen direkten Untersuchungen der mikrobiellen Verhalten innerhalb lückenhaft Lebensräume und realistische kleinen Strömungsverhältnisse ausgeschlossen. Daher hat viel von unseren derzeitigen Kenntnissen in Bezug auf mikrobielle Verhalten in den Ozean von den theoretischen Vorhersagen wurden beschafft. Um neue Informationen über mikrobielle Jagdverhalten in den Ozean wir weichen lithographischen Herstellungstechniken angewendet haben bis 2 mikrofluidischen Systemen, die wir zur Erstellung haben (i) mikroskaligen Nährstoff-Patches mit den Abmessungen und diffusive relevanten Eigenschaften ozeanischer Prozesse und (ii) mikroskaligen entwickeln zu erhalten Wirbel mit Scherraten entsprechend denen in den Ozean erwartet. Diese mikrofluidischen Bauteilen haben eine erste direkte Untersuchung der mikrobiellen Schwimmen und chemotaktische Verhalten innerhalb einer heterogenen und dynamischen seascape gestattet. Der kombinierte Einsatz von Epifluoreszenz und Phasenkontrast-Mikroskopie erlauben den direkten Untersuchungen der physikalischen Abmessungen und diffusive Eigenschaften der Nährstoff-Patches, unter Beachtung der Bevölkerung Ebene aggregative Reaktion, zusätzlich zu den Swimming-Verhalten der einzelnen Mikroben. Diese Experimente haben gezeigt, dass einige Arten von Phytoplankton, heterotrophen Bakterien und phagotrophic Protisten geschickt im Auffinden und die Nutzung diffundierende mikroskaligen Ressource Patches innerhalb sehr kurzer Fristen sind. Wir haben auch gezeigt, dass bis zu Schergeschwindigkeiten moderate sind marinen Bakterien in der Lage, den Fluss und schwimmen durch ihre Umwelt aus eigenem Antrieb zu kämpfen. Doch jenseits einer Schwelle hohe Scher-Ebene, sind die Bakterien in der Scherströmung ausgerichtet sind und weniger in der Lage zu schwimmen, ohne Störung durch den Fluss. Mikrofluidik steht für ein neuartiges und kostengünstigen Ansatz für die Untersuchung aquatischer mikrobielle Ökologie, und aufgrund ihrer Eignung für die genaue realistische Strömungsfelder und Substratgradienten im Mikrobereich, ist ideal für Untersuchungen der mikrobiellen Verhalten bei der kleinsten Skalen der Interaktion. Wir schlagen daher vor, dass Mikrofluidik ein wertvolles Instrument für die Erlangung eines besseren Verständnisses der Ökologie von Mikroorganismen im Meer darstellt.

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

Die Herstellung von mikrofluidischen Kanälen und deren Umsetzung in Experimenten zur Untersuchung der chemotaktischen Jagdverhalten von marinen Mikroben innerhalb eines lückenhaft Nährstoff seascape und das Schwimmverhalten der Bakterien innerhalb Scherströmung beschrieben sind.

Chemotaktische Reaktion der marinen Mikroorganismen zu Micro-Scale Nutrient Layers

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 Aufrufe.  MIT - Massachusetts Institute of Technology. Die Herstellung von mikrofluidischen Kanälen und deren Umsetzung in Experimenten zur Untersuchung der chemotaktischen Jagdverhalten von marinen Mikroben innerhalb eines lückenhaft Nährstoff seascape und das Schwimmverhalten der Bakterien innerhalb Scherströmung beschrieben sind.

Serie: 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.

Schichten von Symbiosis - Visualisierung der Termite Enddarm mikrobiellen Gemeinschaft

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

Forschung

Biologie

Micro-scale Engineering for Cell Biology

Micro-Maßstab Engineering for Cell Biology

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.

In-vivo-Imaging von Deep kortikalen Schichten mit einem MICROPRISMEN

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.

Mit der optokinetischen Reaktion auf visuelle Funktion des Zebrafisch-Studie

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.

Die Quantifizierung der γH2AX Foci in Response to ionisierender Strahlung

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.

Die Messung der Überlebensdauer in<em&gt; Brachionus</em&gt; Rotifers: Synchronisation von Maternal Bedingungen

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.

Hydroponics: ein vielseitiges System Nährstoff Allocation und Pflanzen Reaktionen auf die Nährstoffverfügbarkeit und Belastung durch toxische Elemente zu studieren

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.

Charakterisierung von Verkalkungen Events Verwendung von Live-optische und Elektronenmikroskopie-Techniken in einem Marine-Tubeworm

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.

Detektion von Liganden-aktivierte G-Protein gekoppelten Rezeptor-Internalisierung durch konfokale Mikroskopie

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.

Bewertung von Dictyostelium Discoideum Reaktion auf akute mechanische Stimulation

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