ガラス基板上にCr / Au電​​極を作製したパート1A：ウェットエッチング法 *最高品質のデバイスの場合、製造工程は、クリーンルーム環境で実行する必要がありますまたは層流フードの下になるようほこり、その他の汚染物質は、パターンには影響しません。 <ol><li> 4インチのガラスのスライドによる2インチが置かれて加熱（80℃）ピラニア溶液（5時07分H 2 O 2：H 2 SO 4）汚染物質（特に有機）を削除し、DIでリンスする30分間水と圧縮空気で乾燥。 </li><li> 20 nmのCrおよび200nmのAuを電​​子ビーム蒸着で基板上に堆積されています。 </li><li>シプリー1827ポジ型フォトレジストはspincoater（3000回転、1000回転/秒のランプ、30秒のスピン時間）でスライドガラス上に堆積される。 </li><li>基板は、その後100℃で2分間ソフト焼いています</li><li>マスクのパターンは、206ミリジュール/ cm 2の合計は8.4秒間接触のUV露光でレジストに転送されます。 </li><li>純水リンスに続いて良い撹拌しながら30秒間水（1:3）：フォトレジストは、Microposit MF 351に開発されています。 </li><li> DIの間と後に洗うと良い発展を確保するために顕微鏡で検査した後、基板は、それぞれ15秒と30秒間のAuエッチング液やクロムエッチング液でエッチングされる。 </li></ol>パート1B：代替プロトコル - リフトオフ法<ol><li> 4インチのガラスのスライドによる2インチが置かれて加熱（80℃）ピラニア溶液（5時07分H2O 2：H 2 SO 4）汚染物質（特に有機）を削除し、DI水でリンスする30分間と圧縮空気で乾燥。 </li><li> Futurrex NR - 7 1500 PYネガ型フォトレジストは、基板（2000回転、1000回転/秒のランプ、40秒のスピン時間）にspincoatedした。 </li><li>基板は150℃で1分間焼いたソフトでした</li><li> 21秒（400ミリジュール/ cm 2）のための紫外線照射にお問い合わせください。 </li><li>基板は、その後100℃postbakeステップを完了するには、1分間℃に設定したホットプレート上に置いた。 </li><li>開発はFuturrex RD6、開発者の6秒間実施した。 </li><li> 30nmのCrおよび200nmのAuをし、電子ビーム蒸着で基板上に堆積されています。 </li><li>リフトオフは、金が目に見えて除去し、顕微鏡観察で確認されたまでアセトン超音波浴に基板を配置することによって行われます。 </li></ol>実験的なセットアップ第2部：微小注入して観察<ol><li> PDMSチャネル（製造は別のところで説明）チャンネルが作製電極の上を通過するように直接接着してガラス基板に接続されています。 </li><li>約10 7ミリリットル ポリスチレン微小球は、DI水（0.0002 S / m）またはKCl溶液（0.05 S / m）のいずれかで中断されます。彼らはその後、ミクロスフェアのソリューションでチューブの口を配置し、注射器とコンセントに吸引を適用することにより、注入される。 </li><li>ロードされたデバイスは、顕微鏡ステージ上に配置し、信号発生器に接続されています。 </li><li>観察は顕微鏡で行っている間に周波数設定（1 kHzから1 MHzの）と電圧の設定値（1または2 V）の時間経過が適用されます。 </li></ol>注：これは、高すぎる電圧を上げたり、周波数が低すぎたり、水の電気分解を取得できるようにすることが重要ですが発生します。この発生するために正確な電圧または周波数の設定は、電極の設計に依存しています。私たちの研究室のガイドラインは、8の上方には500 Hzまたは電圧以下の周波数を避けるためにVです。

<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>

このビデオでは、我々はAC動電現象によって引き起こされる粒子と流体操作の挙動を幅広く示している。これらの現象を生成する電極は、製造が容易であり、簡単に他の多くのシステムに統合することができます。これまでに示したように、AC動電学の使用のための多数のアプリケーションがあります。これらのデバイスの多様性だけでなく、操作の迅速な性質は、それらが特に魅力的です。ヘルスケアと他...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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ac electrokinetic phenomena generated by microelectrode structures

AC動電学の分野は​​急速にミクロとラボオンチップアプリケーションに不可欠なナノスケール、上に動的な流体と粒子​​の操作を実行することができるため、成長しています。 AC動電現象は、その液体または懸濁粒子に作用する（誘電体または生物学的な材料で作られたものも含めて）力を生成し、それらの方法1、2を驚くべきに移動させるために電界を使用してください。シングルチャンネル内で、AC動電学はこのようなアクティブなマイクロ混合、粒子分離、粒子の位置とマイクロパターニングなどの多くの本質的なオンチップの操作を行うことができます。単一のデバイスは、単純に印加電圧の周波数や振幅などの動作パラメータを調整することによって、それらの操作のいくつかを達成することができる。適切な電界が容易にマイクロチャネルに統合された微小電極を作成することができます。それはAC動電学の可能性が高い医療診断3-5、環境モニタリング6、国土安全保障7日に大きな影響を持っていることを、この分野で驚異的な成長から明らかである。 一般的には、動作パラメータに一意の依存関係を持つ3つのAC動電現象（AC電気浸透、誘電泳動とAC電熱効果）それぞれがあります。これらの動作パラメータの変化は、このように、粒子または流体の挙動を変える、別の支配的になる一つの現象を引き起こす可能性があります。 それはAC動電学の根底にある複雑な物理学のために、粒子と流体の挙動を予測することは困難である。それは物理学を説明し、粒子と流体の挙動を解明するために本書の目標です。我々の分析はまた、それらを生成する電極構造を作製する方法を説明し、いくつかの一般的なデバイスのデザインを使用して実験観察の広い数を解釈する方法。このビデオの記事では、科学者やエンジニアは、これらの現象を理解する助けとなると、彼らの研​​究ではAC動電学を使い始めることをお勧めすることがあります。

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

AC Electrokinetic Phenomena Generated by Microelectrode Structures

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。

マイクロ電極の構造によって生成されたAC動電現象

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.

コンピュータによって生成された動物モデルの刺激

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

生物学

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.

の蛍光標識ショウジョウバエハートの構造

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.

の可視化線虫（Caenorhabditis elegans）</em親油性生体染色色素、DIIを使用する>キューティクルの構造

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.

3DNAを有する核酸構造の分析と構築

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.

クッパー胞における遺伝子機能と繊毛発生する流体の流れの可視化の分析

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.

イメージングおよび胚ゼブラフィッシュにおける脳血管構造物の3次元再構成

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.

蛍光タンパク質を発現する生きたマウスでのイメージング細胞内の構造物の生体顕微鏡

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.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

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.

イオン選択自己参照微小電極法を用いた細胞外イオンフラックスの測定

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 ...