Fabricating Cr/Au Electrodes on Glass Substrates
Part 1A: Wet Etch Method
*For the highest quality devices, the fabrication process should be performed in a clean room environment or under laminar flow hoods so that dust and other contaminants won't affect the pattern.&nbsp;
<ol>
<li>2-inch by 4-inch Glass slides are placed in a heated (80&deg;C) Piranha solution (5:7 H2O&shy;2:H2SO4) for 30 minutes to remove contaminants (especially organic) and then rinsed in DI water and dried with compressed air. </li>
<li>20 nm Cr and 200 nm Au are deposited onto the substrates with an electron beam evaporator. </li>
<li>Shipley 1827 positive photoresist is deposited on the glass slides with a spincoater (3000 rpm, 1000 rpm/s ramp, 30 second spin time). </li>
<li>Substrates are then soft baked for 2 minutes at 100&deg;C. </li>
<li>The pattern of the mask is transferred to the photoresist with contact UV exposure for 8.4 seconds for a total of 206 mJ/cm2. </li>
<li>The photoresist is developed in Microposit MF 351:Water (1:3) &nbsp;for 30 seconds with good agitation followed by a DI water rinse. </li>
<li>After inspection with a microscope to ensure good development, the substrates are then etched in Au Etchant and Chrome etchant for 15 seconds and 30 seconds respectively with DI washes in between and after. </li>
</ol>
Part 1B: Alternative Protocol - Lift-off Method
<ol>
<li>2-inch by 4-inch Glass slides are placed in a heated (80&deg;C) Piranha solution (5:7 H2O&shy;2:H2SO4) for 30 minutes to remove contaminants (especially organic) and then rinsed in DI water and dried with compressed air. </li>
<li>Futurrex NR-7 1500 PY negative photoresist was spincoated onto the substrate (2000 rpm, 1000 rpm/s ramp, 40 second spin time). </li>
<li>Substrates were soft baked for 1 minute at 150&deg;C. </li>
<li>Contact UV exposure for 21 seconds (400 mJ/cm2). </li>
<li>The substrates were then placed on a hot plate set at 100&deg;C for 1 minute to complete the postbake step. </li>
<li>Development was performed for 6 seconds in Futurrex RD6 developer. </li>
<li>30 nm Cr and 200 nm Au are then deposited onto the substrates with an electron beam evaporator. </li>
<li>Lift-off is performed by placing the substrates in an acetone ultrasonic bath till the gold was visibly removed and confirmed with microscope observation. </li>
</ol>
Experimental Setup
Part 2: Microsphere injection and observation
<ol>
<li>PDMS channels (fabrication described elsewhere) are attached to the glass substrate with direct adhesion so that the channel passes over the fabricated electrodes. </li>
<li>Approximately 107 ml polystyrene microspheres are suspended in either DI water (0.0002 S/m) or a KCl solution (0.05 S/m).&nbsp; They are then injected by placing the tubing inlet in the microsphere solution and applying suction to the outlet with a syringe. </li>
<li>The loaded device is then placed on the microscope stage and connected to a signal generator. </li>
<li>A time course of frequency settings (1 kHz to 1 MHz) and voltage settings (1 or 2 V)&nbsp; are applied while observations are made with the microscope. </li>
</ol>
Note:&nbsp; It is important not to raise the voltage too high or allow the frequency to get too low or electrolysis of water will occur.&nbsp; The exact voltage or frequency settings for this to occur are dependant on the electrode design.&nbsp; Our lab guidelines are to avoid frequencies below 500 Hz or voltages above 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 this video, we have shown a wide variety of particle and fluid manipulation behaviors caused by AC electrokinetic phenomena. The electrodes that generate these phenomena are easy to fabricate and can be easily integrated into many other systems. As we have shown, there are numerous applications for the use of AC electrokinetics. The versatility of these devices, as well as the rapid nature of manipulation, makes them particularly attractive. As healthcare and other industries begin to embrace lab-on-a-chip systems, we...

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		<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>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>
		</table>
		</div>

ac electrokinetic phenomena generated by microelectrode structures

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, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

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

AC Electrokinetic Phenomena Generated by Microelectrode Structures

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

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 ビュー.  Drexel University. マイクロナノスケールは、より現実のようになっている可能にした技術で流体と懸濁粒子を操作すると、AC動電学のように、開発を進めています。ここで、我々は、これらのデバイスを作製する方法および実験観察を解釈する方法、AC動電学の背後にある物理学を議論する。

ビデオ: 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.

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

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.

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

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

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.

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.

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.

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.

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.

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.

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