Fabbricazione di Cr / Au Elettrodi su substrati di vetro Parte 1A: Wet Metodo Etch * Per i dispositivi di alta qualità, il processo di fabbricazione deve essere eseguita in un ambiente sterile o sotto cappe a flusso laminare in modo che polvere e altri contaminanti non influirà sul modello. <ol><li> 2-pollici da 4 pollici vetrini sono posti in una riscaldata (80 ° C) Piranha soluzione (5:7 H 2 O 2: H 2 SO 4) per 30 minuti per rimuovere i contaminanti (soprattutto organici) e poi risciacquati in DI acqua e asciugati con aria compressa. </li><li> Cr 20 nm e 200 nm di Au sono depositati su substrati con un evaporatore a fascio di elettroni. </li><li> Shipley 1827 photoresist positivo si deposita sui vetrini con un spincoater (3000 rpm, 1000 rpm / s rampa, 30 tempo di rotazione secondo). </li><li> Substrati sono poi cotto morbido per 2 minuti a 100 ° C. </li><li> Il modello della maschera è trasferito al photoresist con l&#39;esposizione ai raggi UV contatto per 8,4 secondi per un totale di 206 mJ / cm 2. </li><li> Il photoresist è sviluppato in Microposit MF 351: acqua (1:3) per 30 secondi con una buona agitazione seguita da un risciacquo acqua deionizzata. </li><li> Dopo l&#39;ispezione al microscopio per assicurare un buon sviluppo, i substrati vengono poi incise in Au Mordenzante e Chrome mordenzante per 15 secondi e 30 secondi rispettivamente con DI lava in mezzo e dopo. </li></ol> Parte 1B: Protocollo alternative - Lift-off Metodo <ol><li> 2-pollici da 4 pollici vetrini sono posti in una riscaldata (80 ° C) Piranha soluzione (5:7 H2O 2: H 2 SO 4) per 30 minuti per rimuovere i contaminanti (soprattutto organici) e poi risciacquati in acqua deionizzata e asciugati con aria compressa. </li><li> Futurrex NR-7 1500 PY photoresist negativo è stato spincoated sul substrato (2000 rpm, 1000 rpm / s rampa, 40 tempo di rotazione secondo). </li><li> Substrati erano morbidi al forno per 1 minuto a 150 ° C. </li><li> Contatta esposizione ai raggi UV per 21 secondi (400 mJ / cm 2). </li><li> I supporti sono stati poi posto su un piatto caldo a 100 ° C per 1 minuto per completare il passaggio termoindurimento. </li><li> Sviluppo è stata effettuata per 6 secondi in sviluppatore Futurrex RD6. </li><li> Cr 30 nm e 200 nm di Au vengono depositati su substrati con un evaporatore a fascio di elettroni. </li><li> Decollo viene eseguito ponendo i substrati in un bagno di acetone fino ad ultrasuoni l&#39;oro era visibilmente rimosso e confermata con osservazione al microscopio. </li></ol> Setup sperimentale Parte 2: iniezione di microsfere e osservazione <ol><li> PDMS canali (fabbricazione descritto altrove) sono attaccati al substrato di vetro con adesione diretta in modo che il canale passa sopra gli elettrodi fabbricato. </li><li> Circa 10 7 ml polistirolo microsfere sono sospese in acqua deionizzata o (0,0002 S / m) o una soluzione di KCl (0,05 S / m). Essi vengono poi iniettate posizionando il tubo di aspirazione nella soluzione di microsfere e applicando aspirazione alla presa con una siringa. </li><li> Il dispositivo caricato viene posto sul palco microscopio e collegata ad un generatore di segnale. </li><li> Una durata di impostazioni di frequenza (1 kHz a 1 MHz) e le impostazioni di tensione (1 o 2 V) vengono applicate, mentre le osservazioni sono fatte con il microscopio. </li></ol> Nota: è importante non alzare il voltaggio troppo alto o consentire la frequenza troppo bassa o elettrolisi dell&#39;acqua si verificherà. Le impostazioni di voltaggio o la frequenza esatta per questo si verifichi dipendono dalla progettazione degli elettrodi. Le nostre linee guida di laboratorio devono evitare frequenze inferiori a 500 Hz o tensioni maggiori di 8 V.

<ol>
	<li>Ramos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AC+Electrokinetics:+a+review+of+forces+in+microelectrode+structures.">AC Electrokinetics: a review of forces in microelectrode structures.</a> Journal of Physics D: Applied Physics. 31, 2338-2353 (1998).</li><li>Morgan, H. y. w. e. l., Green, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AC+Electrokinetics:+colloids+and+nanoparticles.">AC Electrokinetics: colloids and nanoparticles.</a> , (2002).</li><li>Toner, M., Irimia, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-on-a-chip.">Blood-on-a-chip.</a> Annual Review of Biomedical Engineering. 2005, 77-103 (2005).</li><li>Ahn, C. H., Choi, J. -. W., Beaucage, G., Nevin, J. H., Lee, J. -. B., Puntambekar, A., Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposable+smart+lab+on+a+chip+for+point+of+care+clinical+diagnostics.">Disposable smart lab on a chip for point of care clinical diagnostics.</a> 282, 399-401 (1998).</li><li>Vespoorte, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+chips+for+clinical+and+forensic+analysis.">Microfluidic chips for clinical and forensic analysis.</a> Electrophoresis. 23, 677-712 (2002).</li><li>Rajaraman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+low+cost+microfabrication+technologies+toward+realization+of+devices+for+dielectrophoretic+manipulation+of+particles+and+nanowires.">Rapid, low cost microfabrication technologies toward realization of devices for dielectrophoretic manipulation of particles and nanowires.</a> Sensors and Actuators B: Chemical. 114, 392-401 (2006).</li><li>Ali, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-a-chip+for+terrorist+weapons+management.">Lab-on-a-chip for terrorist weapons management.</a> Measurement and Control. 38, 87-91 (2005).</li><li>Voldman, J. o. e. l., Rosenthal, A. d. a. m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectrophoretic+Traps+for+Single-particle+Patterning.">Dielectrophoretic Traps for Single-particle Patterning.</a> Biophysical Journal. 88, 2193-2205 (2005).</li><li>Ramachandran, T. R., Baur, C., Bugacov, A., Madhukar, A., Koel, B. E., Requicha, A., Gazen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+controlled+manipulation+of+nanometer-sized+particles+using+the+non-contact+atomic+force+microscope.">Direct and controlled manipulation of nanometer-sized particles using the non-contact atomic force microscope.</a> Nanotechnology. 9, 237-245 (1998).</li><li>Sigurdson, M. a. r. i. n., Wang, D., Meinhart, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrothermal+stirring+for+heterogeneous+immunoassays.">Electrothermal stirring for heterogeneous immunoassays.</a> Lab Chip. 5, 1366-1373 (2005).</li><li>Urbanski, J. o. h. n. . P. a. u. l., Levitan, J. e. r. e. m. y. A., Bazant, M. a. r. t. i. n. Z., Thorsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+ac+electro-osmotic+micropumps+with+non-planar+electrodes.">Fast ac electro-osmotic micropumps with non-planar electrodes.</a> Appl. Phys. Lett. 89, 143508 (2006).</li><li>Fatoyinbo, H. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+dielectrophoretic+quartz+crystal+microbalance+(DEP-QCM)+device+for+rapid+biosensing+applications.">An integrated dielectrophoretic quartz crystal microbalance (DEP-QCM) device for rapid biosensing applications.</a> Biosens Bioelectron. 23, 225-232 (2007).</li></ol>

In questo video, abbiamo dimostrato una grande varietà di comportamenti di manipolazione di particelle e fluidi causata da fenomeni AC elettrocinetici. Gli elettrodi che generano questi fenomeni sono facili da fabbricare e può essere facilmente integrato in molti altri sistemi. Come abbiamo dimostrato, ci sono numerose applicazioni per l&#39;utilizzo di elettrocinesi AC. La versatilità di questi dispositivi, così come la natura rapida della manipolazione, le rende particolarmente attraente. Le industrie sanitarie e gli altri cominciano ...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>2" by 4" Pyrex Glass Slide</td>
<td>Substrate</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Pyrex 7740</td>
</tr>
<tr>
<td>chrome mask</td>
<td>material</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>This photomask will have the microelectrode patterns on them and can be ordered from a variety of microfabrication centers.</td>
</tr>
<tr>
<td>PDMS Microchannels</td>
<td>material</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>These may be fabricated and used in-house or a simple microscope slide will suffice.</td>
</tr>
<tr>
<td>Hydrogen Peroxide 30%</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>7722-84-1</td>
<td>Certified ACS, Fisher Scientific</td>
</tr>
<tr>
<td>Sulfuric Acid</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>A300-212</td>
<td>Certified ACS Plus</td>
</tr>
<tr>
<td>Acetone Electronic Grade</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>A946-4</td>
<td>&nbsp;</td>
<tr>
<td>Shipley 1827 Positive Photoresist</td>
<td>Reagent</td>
<td>Microchem Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Shipley 351 Developer</td>
<td>Reagent</td>
<td>Microchem Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gold Etchant</td>
<td>Reagent</td>
<td>Transene Company, Inc.</td>
<td>Type TFA</td>
<td>&nbsp;</td>
<tr>
<td>Chrome Photomask Etchant</td>
<td>Reagent</td>
<td>Cyantek Corporation</td>
<td>CR-7S</td>
<td>&nbsp;</td>
<tr>
<td>NR-7 1500 PY Negative Resist</td>
<td>Reagent</td>
<td>Futurrex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RD6 Developer</td>
<td>Reagent</td>
<td>Futurrex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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ac electrokinetic phenomena generated by microelectrode structures

Il campo di elettrocinesi CA è in rapida crescita grazie alla sua capacità di eseguire fluidodinamica e la manipolazione di particelle sulla micro-e nano-scala, che è essenziale per Lab-on-a-Chip applicazioni. Fenomeni AC elettrocinetici utilizzare i campi elettrici di generare forze che agiscono sui liquidi o particelle sospese (compresi quelli fatti di dielettrico o materiale biologico) e causano loro di muoversi in modi sorprendenti 1, 2. All&#39;interno di un singolo canale, elettrocinesi AC può realizzare molti essenziali on-chip operazioni come attivo il posizionamento di particelle micro-miscelazione, separazione delle particelle, e micro-picchiettare. Un unico dispositivo può compiere diverse di tali operazioni con la semplice regolazione dei parametri operativi, come la frequenza o l&#39;ampiezza della tensione applicata. Adatto campi elettrici possono essere facilmente create da micro-elettrodi integrati in microcanali. E &#39;chiaro dalla crescita enorme in questo campo che elettrocinesi AC probabilmente avrà un effetto profondo sulla diagnostica sanitaria 3-5, monitoraggio ambientale 6 e homeland security 7. In generale, ci sono tre fenomeni elettrocinetici CA (AC elettroosmosi, dielettroforesi ed effetto AC elettrotermica) ciascuno con dipendenze unico sui parametri di funzionamento. Un cambiamento di questi parametri di funzionamento può causare uno dei fenomeni a diventare dominante su un&#39;altra, cambiando così la particella o il comportamento dei fluidi. E &#39;difficile prevedere il comportamento delle particelle e liquidi a causa della fisica che sono alla base complicata elettrocinesi AC. E &#39;l&#39;obiettivo di questa pubblicazione per spiegare la fisica delle particelle e di spiegare e il comportamento dei fluidi. La nostra analisi riguarda anche il modo di fabbricare le strutture elettrodo che li generano, e come interpretare un ampio numero di osservazioni sperimentali utilizzando diversi disegni popolare dispositivo. In questo articolo il video aiuterà gli scienziati e gli ingegneri capire questi fenomeni e possono incoraggiarli a iniziare a utilizzare elettrocinesi AC nella loro ricerca.

Watch this Scientific Journal Video about AC Electrokinetic Phenomena Generated by Microelectrode Structures at JoVE.com

AC Electrokinetic Phenomena Generated by Microelectrode Structures

Manipolare fluidi e particelle in sospensione nelle tecnologie micro-e nano-scala è sempre più di una realtà come abilitazione, come elettrocinesi AC, continuare a sviluppare. Qui, si discute la fisica dietro elettrocinesi AC, come fabbricare questi dispositivi e come interpretare le osservazioni sperimentali.

Fenomeni AC elettrocinetico Generato da strutture microelettrodo

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

ac-electrokinetic-phenomena-generated-by-microelectrode-structures

Research

JoVE Journal

Biology

11.4K Views.  Drexel University. Manipolare fluidi e particelle in sospensione nelle tecnologie micro-e nano-scala è sempre più di una realtà come abilitazione, come elettrocinesi AC, continuare a sviluppare. Qui, si discute la fisica dietro elettrocinesi AC, come fabbricare questi dispositivi e come interpretare le osservazioni sperimentali.

Video: AC Electrokinetic Phenomena Generated by Microelectrode Structures

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated modello animale Stimoli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Ricerca

Biologia

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Etichette fluorescenti di Drosophila Cuore Strutture

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Visualizzazione di Caenorhabditis Elegans Strutture cuticola Utilizzando il Dye lipofili Vital, Dii

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

Analisi e costruzione di strutture acidi nucleici con 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

Analisi della funzione del gene e visualizzazione di Cilia-Generated flusso di fluido in vescicole di Kupffer

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Imaging 3D e ricostruzione di strutture cerebrovascolari in Zebrafish embrionali

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Intravitale Microscopia per le strutture Imaging subcellulari in topi vivi Esprimere proteine ​​fluorescenti

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Misurazione della extracellulare Ion Flussi Utilizzando il ionoselettivo autoreferenziale microelettrodo Tecnica

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...