Vorremmo ringraziare Microsystems Technology Laboratories al MIT per averci permesso di parte pellicola di questo video nella struttura camera pulita.

Preparazione 1. Creare una maschera <blockquote> Utilizzando un software CAD, la progettazione del canale per la stampa ad alta risoluzione su un lucido. Questa sarà la &quot;maschera&quot;. </blockquote> Nella camera bianca: 2. Pulire e cuocere la cialda In primo luogo, spruzzare il wafer con acetone, poi rapidamente con metanolo, poi con isopropanolo. Infine, asciugare le fette che utilizzano azoto. Cuocere i wafer in forno (130 ° C) per 5 min. 3. Rivestimento del wafer Posizionare il wafer al centro dello spin-coating macchina. Versare photoresist (SU-8) dalla bottiglia sul wafer. Lasciate che il SU-8 di flusso e rilassarsi per circa 10 s. Accendere lo spin-verniciatore e la sua velocità rampa salita da 0 a 500 rpm per 5 s; tenere a 500 rpm per 10 s; rampa fino alla velocità finale di oltre 10 s e mantenere a velocità finale per 30 s. La velocità finale dipende dallo spessore del rivestimento mirati e il SU-8 utilizzati. I dettagli sono disponibili all&#39;indirizzo <a href="http://www.microchem.com/">http://www.microchem.com/</a> 4. Soft-cuocere Dopo il rivestimento del wafer, cuocere prima a 65 ° C e poi a 95 ° C. Il tempo di cottura varia a seconda dello spessore e del tipo di mirati photoresist utilizzato. Poi, lasciare che il wafer a temperatura ambiente per almeno 5 minuti. 5. Esposizione Collocare la maschera sulla parte superiore del wafer e di esporre il wafer alla luce UV per il tempo indicato nel SU-8 manuale. 6. Post-esposizione cuocere Cuocere il wafer a 65 ° C e poi 95 ° C in seguito alla SU-8 manuale di istruzioni. 7. Sviluppare la wafer per ottenere il &quot;master&quot; (muffe) Preparare un bicchiere riempito con lo sviluppatore (PMMA). Immergere il wafer nel becher mentre molto dolcemente oscillanti il ​​bicchiere fino a quando la parte non esposta del photoresist viene lavato via. Nel nostro laboratorio: 8. Preparare PDMS e versarlo sul wafer Mescolare il PDMS con il suo catalizzatore nel rapporto 10:1 in una tazza. Mescolare e mescolare in maniera omogenea: questo genererà un sacco di bolle e di rendere la miscela aspetto opaco. Versare il composto sul &quot;master&quot;. 9. De-bolla nel vuoto da camera Per rimuovere le bolle, ha messo la miscela master e PDMS che si copre in una camera a vuoto fino a quando tutte le bolle sono scomparse. 10. Cottura in forno Cuocere in forno per almeno 12 ore in un forno a 65 ° C per indurire il PDMS. 11. Perforare Staccare il PDMS dal master e perforare per ingressi e le uscite dei canali. In camera bianca (non mostrato) 12. Plasma incollaggio I canali sono legati ad una lastra di vetro dopo aver trattato sia il livello PDMS e il vetrino con plasma di ossigeno per 1 min. Esperimenti: Exp # 1: Indagare la risposta chemiotattica dei microbi marini a strati nutrienti micro-scala 1) Impostare l&#39;esperimento <ol><li> Aggiungi organismi e substrati di siringhe di vetro </li><li> Luogo canale microfluidica sul palco microscopio e allegare tubi di ingressi appropriati e le prese </li><li> Collegare il tubo di rifiuti serbatoio. Assicurarsi tubi è completamente immerso nel liquido nel serbatoio dei rifiuti per evitare oscillazioni di pressione </li><li> Siringhe posto sulla pompa a siringa e collegare alle valvole e tubi </li><li> Impostare microscopio: le condizioni di luce, ingrandimento, ecc </li><li> Focus sulla posizione appropriata nel canale </li><li> De-bolla canale usando più grandi siringa riempita con acqua di mare artificiale </li><li> Impostare la velocità di flusso adeguato di pompa a siringa. In questo caso 2 ml / min, che corrisponde ad una velocità di flusso medio di -1 220 micron s nel canale </li></ol> 2) Eseguire l&#39;esperimento <ol><li> Avvio pompa a siringa per stabilire un gradiente di nutrienti nel canale </li><li> Una volta che il flusso si è stabilizzata e una banda di sostanze nutritive è sviluppato, fermare il flusso della pompa siringa e iniziare a registrare il tempo da questo punto </li><li> Banda nutrienti comincia a diffondere lateralmente </li><li> A intervalli di tempo regolari, utilizzare software di analisi dell&#39;immagine per registrare le sequenze di fotogrammi per creare &#39;film&#39; </li><li> Discriminare gli organismi nuoto prendendo tempo-differenza immagini tra due fotogrammi successivi, in modo che solo gli oggetti in movimento sono ora visualizzate, che ci permette di differenziare le cellule mobili da non-particelle in movimento e il rumore di fondo </li><li> Prendete i film per determinare le posizioni di cellule nel canale con riferimento al posizione della patch nutrienti </li><li> Registrazione di filmati ad intervalli regolari di 10-20 minuti per analizzare le posizioni e modelli di nuoto di organismi </li><li> Usando il software di analisi dell&#39;immagine per sovrapporre le posizioni degli organismi a diversi frame (assegnando ad ogni pixel l&#39;intensità massima della luce registrata in quel pixel per tutta la durata del film), possiamo ottenere informazioni traiettoria per le cellule di nuoto </li></ol> Exp # 2: Indagare gli effetti del taglio sui batteri marini nuotare in un vortexZ <ol><li> Utilizzando la geometria canale diverso, siamo in grado di osservare il comportamento dei batteri nuotare in un vortice a diversi tassi di taglio </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>

La comprensione di come i microbi marini interagiscono con i loro chimica locale e l&#39;ambiente fisico è fondamentale per una percezione più completa e precisa del ruolo dei microrganismi planctonici nel nutrienti oceani e cicli di carbonio (Azam e Malfatti 2007). Tuttavia, a causa delle piccole scaglie (mm &lt;) su cui molte interazioni microbiche si svolgono, limitazioni tecniche hanno impedito esami dettagliata del comportamento microbica all&#39;interno dell&#39;eterogeneo bio-fisico-chimiche del paesaggio previsto per essere sperim...

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<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>
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<td>SU8-2100</td>
<td>Photoresist</td>
<td>MicroChem, Newton, MA, USA</td>
<td>&nbsp;</td>
<td>www.microchem.com
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<td>Nikon Eclipse TE2000-E inverted microscope</td>
<td>Microscope</td>
<td>Nikon, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<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>
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<td>Syringes (Luer-Lok Tip)</td>
<td>Tool</td>
<td>BD, Franklin Lakes, NJ, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<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>
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<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>
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chemotactic response of marine micro-organisms to micro-scale nutrient layers

Il grado con cui i microbi planctonici possono sfruttare le patch risorsa microscala avrà notevoli implicazioni per trophodynamics oceaniche e flussi biogeochimici. Tuttavia, per sfruttare le patch nutrienti nell&#39;oceano, microbi nuoto devono superare le influenze delle forze fisiche inclusa la diffusione molecolare e turbolenta di taglio, che limiterà la disponibilità di patch e la capacità dei batteri di individuarli. Fino a poco tempo, le limitazioni metodologiche hanno precluso esami diretta del comportamento microbica all&#39;interno di habitat macchia di leopardo e realistico di piccole dimensioni condizioni di flusso. Quindi, gran parte delle nostre attuali conoscenze sul comportamento microbica negli oceani è stata ottenuta da previsioni teoriche. Per ottenere nuove informazioni sul comportamento alimentare microbica in mare abbiamo applicato morbido tecniche di fabbricazione litografica per sviluppare 2 dispositivi microfluidica, che abbiamo usato per creare (i) microscala patch nutrienti con dimensioni e caratteristiche diffusive rilevanti per i processi oceanici e (ii) microscala vortici, con tassi di taglio corrispondenti a quelle previste nel mare. Questi dispositivi microfluidici hanno permesso un primo esame diretto di nuoto microbica e del comportamento chemiotattici all&#39;interno di un paesaggio marino eterogeneo e dinamico. L&#39;uso combinato di epifluorescenza e microscopio a contrasto di fase permette esami diretta delle dimensioni fisiche e le caratteristiche diffusive di patch nutrienti, nel rispetto della popolazione risposta a livello aggregativo, in aggiunta al comportamento di nuoto di microbi individuali. Questi esperimenti hanno rivelato che alcune specie di fitoplancton, batteri e protisti eterotrofi phagotrophic sono abili a individuare e sfruttare le risorse microscala diffondere le patch in tempi molto brevi. Abbiamo inoltre dimostrato che fino a moderati tassi di taglio, batteri marini in grado di combattere il flusso e nuotare attraverso il loro ambiente a loro spontanea volontà. Tuttavia, al di là di un alto livello di soglia di taglio, i batteri sono allineati nel flusso di taglio e sono meno in grado di nuotare senza disturbi dal flusso. Microfluidica rappresenta un approccio nuovo e poco costoso per lo studio dell&#39;ecologia microbica acquatica, e grazie alla sua idoneità con precisione la creazione di campi di flusso e gradienti di substrato realistico alla microscala, è perfettamente applicabile agli esami di comportamento microbica a scale più piccole di interazione. Pertanto suggeriamo che microfluidica rappresenta uno strumento prezioso per ottenere una migliore comprensione dell&#39;ecologia della microrganismi nell&#39;oceano.

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 realizzazione di canali di microfluidica e la loro attuazione in esperimenti per studiare il comportamento chemiotattica foraggiamento dei microbi marini all&#39;interno di un paesaggio marino irregolare dei nutrienti e il comportamento di nuoto di batteri all&#39;interno del flusso di taglio sono descritti.

Risposta chemiotattica di microrganismi marini a livelli di nutrienti Micro-Scale

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 Views.  MIT - Massachusetts Institute of Technology. La realizzazione di canali di microfluidica e la loro attuazione in esperimenti per studiare il comportamento chemiotattica foraggiamento dei microbi marini all'interno di un paesaggio marino irregolare dei nutrienti e il comportamento di nuoto di batteri all'interno del flusso di taglio sono descritti.

Video: 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.

Livelli di simbiosi - Visualizzazione della Comunità hindgut Termite microbica

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

Ricerca

Biologia

Micro-scale Engineering for Cell Biology

Micro-scala di Ingegneria di biologia cellulare

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.

Imaging in vivo di Deep strati corticali usando un microprismi

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.

Utilizzando la risposta optocinetico per studiare la funzione visiva del pesce zebra

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.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

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.

Misurazione del Tempo di sopravvivenza in<em&gt; Brachionus</em&gt; Rotiferi: Sincronizzazione di materni Condizioni

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: un sistema versatile per studiare nutrienti allocazione e vegetali risposte a disponibilità di nutrienti e l&#39;esposizione a elementi tossici

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.

Caratterizzazione di calcificazione eventi utilizzando dal vivo ottica ed elettronica Microscopia tecniche in 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.

Rilevamento di Ligand-activated proteina G-recettore accoppiato internalizzazione da microscopia 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.

Valutazione della risposta di Dictyostelium discoideum alla stimolazione meccanica acuta

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