유리 기판에 CR / 오 전극을 Fabricating 부품 1A : 습식 엣지 방법 * 최고 품질의 장치, 제조 공정은 클린룸 환경에서 수행되어야하거나 층류 흐름 후드 아래에 있도록 먼지 및 기타 오염 물질의 패턴에 영향을 미치지 않습니다. <ol><li> 4 인치 유리 슬라이드에 의해 2 인치가에 배치하는 온수 (80 ° C) 피라 냐가 솔루션 (5시 7분 H 2 O 2 : H 2 SO 4) 오염 물질을 (특히 유기)를 제거 후 DI에 씻어서 30 분 물과 압축 공기 건조. </li><li> 20 nm의 피크와 200 nm의 오는 전자빔 증발기와 기판에 입금됩니다. </li><li> 시플리 1,827 긍정적인 포토 레지스트는 spincoater (3000 RPM 1000 RPM / s의 램프, 30초 회전 시간)와 함께 유리 슬라이드에 입금됩니다. </li><li> 기판은 다음 100 ° C. 2 분 동안 부드럽게 구운 아르 </li><li> 마스크의 패턴은 206 MJ / cm 2 총 8.4 초 동안 연락 자외선 노출과 포토 레지스트로 전송됩니다. </li><li> DI 워터 헹굼 다음 잘 교반 30 초 동안 물 (1시 3분) : 포토 레지스트는 Microposit MF 351으로 개발되고 있습니다. </li><li> DI는 사이 후의 세척과 좋은 발전을 보장하기 위해 현미경으로 검사 후, 기판은 다음 각각 15초 30 초 오 에칭제과 크롬 에칭에 새겨져 있습니다. </li></ol> 부품 1B : 대체 프로토콜 - 리프트 - 오프 방법 <ol><li> 4 인치 유리 슬라이드에 의해 2 인치가에 배치하는 온수 (80 ° C) 피라 냐가 솔루션 (5시 7분 H2O 2 : H 2 SO 4) 오염 물질을 (특히 유기)를 제거하고 디 물에 씻어서 30 분 및 압축 공기 건조. </li><li> Futurrex NR - 7 1,500 PY 부정적인 포토 레지스트는 (2000 RPM 1000 RPM / s의 램프, 40초 회전 시간) 기판에 spincoated했다. </li><li> 기판은 150 ° C.에 1 분 구운 부드러운되었습니다 </li><li> 21초 (400 MJ / cm 2) 자외선 노출에 문의하십시오. </li><li> 기판은 다음 ° C는 1 분 postbake 단계를 완료하기 위해 100로 설정 뜨거운 접시에 놓여 있었다. </li><li> 개발 Futurrex RD6 개발자 6 초 동안 수행되었다. </li><li> 30 nm의 피크와 200 nm의 오 그러면 전자빔 증발기와 기판에 입금됩니다. </li><li> 이륙은 금괴가 가시 제거하고 현미경 관찰과 확인 살이 아세톤 초음파 욕조에서 기판을 배치하여 수행됩니다. </li></ol> 실험 설정 2 부 : Microsphere 주입 및 관찰 <ol><li> 채널 가공 전극 통과되도록 PDMS 채널 (제조는 다른 설명)가 직접 접착력으로 유리 기판에 붙어 있습니다. </li><li> 약 10 7 ML 폴리스티렌 microspheres은 DI 워터 (0.0002 S / M) 또는 KCl 용액 (0.05 S / M) 중 하나에서 일시 중지됩니다. 그들은 다음 microsphere 솔루션에서 튜브의 입구를 삽입하고 주사기로 콘센트에 흡입을 적용하여 주입합니다. </li><li> 로드된 장치는 다음 현미경 스테이지에 배치하고 신호 발생기에 연결됩니다. </li><li> 관찰은 현미경으로 만들어집니다 동안 주파수 설정 (1 kHz에서 1 MHz 이상) 및 전압 설정 (1 또는 2 V)의 시간 과정은 적용됩니다. </li></ol> 참고 : 너무 높은 전압을 늘리거나 주파수가 발생합니다 너무 낮게 또는 물의 전기 분해로 얻을 수 있습니다 않을 것이 중요합니다. 이 발생하는 정확한 전압 또는 주파수 설정은 전극 설계에 따라 달라집니다. 우리 연구실 지침 8 위 500 Hz에서 전압 또는 아래의 주파수를 피하기 위해 아르 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>

이 비디오에서는, 우리는 AC 계면 현상에 의한 입자와 유체 조작 행동의 다양한 나타났습니다. 이러한 현상을 생성하는 전극은 조작하기 쉽고 쉽게 다른 많은 시스템에 통합될 수 있습니다. 우리가 표시된 것처럼, AC 동전 기학의 사용에 대한 여러 응용 프로그램이 있습니다. 이러한 장치의 융통성뿐만 아니라 조작의 급속한 자연, 그들은 특히 매력적 수 있습니다. 건강 관리 및 기타 산업 연구실 - 온 - 칩 시스템?...

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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

AC 동전 기학 분야는 빠르게 인해의 마이크로 및 나노 스케일, 랩 - 온 - 어 - 칩 응용 프로그램을위한 필수되는 역동적인 유체 및 입자 조작을 수행하는 능력으로 성장하고있다. AC 계면 현상은 유체 또는 중지 입자 (유전체 또는 생물 학적 물질로 만들어져 포함)와 행동은 그들이 가지 방법 1, 2 놀라운으로 이동하는 원인이되는 힘을 생성하는 전기 필드를 사용합니다. 하나의 채널 내에서, AC 동전 기학는 활성 마이크로 혼합, 입자 분리, 입자 위치 및 마이크로 pattering 많은 필수 온 - 칩 작업을 수행할 수 있습니다. 하나의 장치는 단순히 같은 주파수 또는 적용 전압의 진폭과 같은 작동 매개 변수를 조정하여 이러한 작업을 여러를 달성할 수 있습니다. 적합 전기 필드는 쉽게 microchannels 통합 마이크로 전극에 의해 만들 수 있습니다. 그것은 AC 동전 기학 가능성이 의료 진단 3-5, 환경 모니터링 6 국토 안보부 7 일에 깊은 영향을 미칠 것이라고이 분야에서 엄청난 성장에서 분명하다. 일반적으로 세 AC 계면 현상 (AC electroosmosis, dielectrophoresis와 AC electrothermal 효과) 운영 매개 변수에 고유한 종속성이 각각있다. 이러한 운영 매개 변수의 변경은 따라서 입자 또는 액체의 행동을 바꾸고, 다른 이상 지배되는 하나의 현상을 일으킬 수 있습니다. 그것은 AC 동전 기학을 기초 복잡한 물리학으로 인해 입자와 유체의 행동을 예측하기가 어렵습니다. 그것은 물리학을 설명하고 입자와 유체 행동을 명료하게하다이 간행물의 목표입니다. 우리의 분석은 또한 그들을 생성하는 전극 구조를 조작하는 방법을 다루고, 여러 인기있는 장치 설계를 사용하여 실험 관찰 다양한 숫자를 해석하는 방법. 이 동영상이 문서는 과학자와 엔지니어가 이러한 현상을 이해하고 자신의 연구에 AC 동전 기학을 사용하여 시작하는 것이 좋습니다 수도 있습니다 도움이 될 것입니다.

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

AC Electrokinetic Phenomena Generated by Microelectrode Structures

마이크로 및 나노 스케일 더 현실로되어 가고 있도록 기술 및 정지 유체 입자를 조작하는 것은, AC 동전 기학 같은 개발을 계속합니다. 여기, 우리는 어떻게 실험 관찰 해석하는 이러한 장치와를 조작하는 방법, AC 동전 기학 뒤에 물리학에 대해 설명합니다.

Microelectrode 구조에 의해 발생 AC 계면 현상

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 조회수.  Drexel University. 마이크로 및 나노 스케일 더 현실로되어 가고 있도록 기술 및 정지 유체 입자를 조작하는 것은, AC 동전 기학 같은 개발을 계속합니다. 여기, 우리는 어떻게 실험 관찰 해석하는 이러한 장치와를 조작하는 방법, AC 동전 기학 뒤에 물리학에 대해 설명합니다.

비디오: 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.

컴퓨터가 만든 동물 모델 자극

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

연구

생물학

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.

의 형광 라벨링 Drosophila 심장 구조

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.

이중 - 공진 코히어런트 반 스톡스 래맨 산란 (자동차)와 생물 학적 구조의 차동 영상

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.

의 시각화 Caenorhabditis Elegans</emLipophilic 바이탈 염료, DiI를 사용하기> 표피 구조

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.

3DNA과 핵산의 구조를 분석하고 구축

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.

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.

Kupffer의 소포의 유전자 기능 및 속눈썹 생성 된 유체 흐름의 시각화 분석

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.

이미징 및 배아 제브라 피쉬의 뇌 혈관 구조의 3 차원 재구성

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.

형광 단백질을 표현 라이브 마우스의 이미징 세포 이하의 구조물의 intravital 현미경

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.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

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