由美国国立卫生研究院授予HL89764，美国国家科学基金会赠款0546012，0730503和0725528提供资金支持。

A.生物素化的DNA 质粒DNA共价键所述的制造商（Mirus生物，麦迪逊，威斯康星州），但规模有〜1-2生物素的DNA标签的标签与鸟嘌呤特定的生物素生物素。质粒DNA（pEGFP - C1的，4.9 KB，Clontech公司，美国加州Mountain View），是标示在本议定书。 <ol><li> DNase的自由和无RNase的TE缓冲液（分子生物学级质量）水溶解成所需PDNA金额作出1μg/μL的DNA溶液。 </li><li>使用以下的反应混合物进行标记反应。添加标签的IT试剂去年。 </li></ol> 100μgDNA的反应： <table border="1"><tbody><tr><td> DNase的自由和无RNase水</td><td> 75微升</td></tr><tr><td> 10X标记缓冲液A </td><td> 20微升</td></tr><tr><td> 1μg/μL的DNA </td><td> 100μL， </td></tr><tr><td> 标签上试剂</td><td> 5微升</td></tr><tr><td> 总成交量 </td><td> 200微升</td></tr></tbody></table><ol start="3"><li>孵育反应在37 ° C为1小时。 </li><li>下列标准协议的乙醇或异丙醇沉淀净化标记的样本。 </li></ol> 注：凝胶过滤的列可能会导致高的紫外吸收或荧光背景，这可能会影响DNA定量荧光特性。 注：生物素的水平可能由HABA为基础的测试来决定。 B. Cy5的阳离子聚合物标签壳聚糖（390 kDa的，83.5％脱乙酰，万顺，雷德蒙，华盛顿州）被用来作为一种模式在这项研究中的阳离子聚合物。壳聚糖聚合物骨架上的自由伯胺被打成用Cy5 - NHS（Amersham公司生物科技公司，新泽西州皮斯卡塔韦）。 <ol><li>为了方便完成Cy5的染料的共轭，所需金额计算Cy5的- NHS的Cy5的伯胺的摩尔比为1：200。 </li><li> 〜6.5的NaOH除了调整壳聚糖溶液的pH值（在25mm的醋酸缓冲液）。注意NHS的反应是在碱性条件更有效率，但这里壳聚糖的溶解度限制了工作的pH值范围。 </li><li>边搅拌，慢慢加入Cy5的国民保健服务（1毫克/毫升二甲基亚砜）的计算量的壳聚糖溶液在下降，下降的方式。 </li><li>在黑暗中的混合物在室温下搅拌过夜。 </li><li>净化，对在黑暗的房间温度在1％的醋酸缓冲液透析与10K MWCO幻灯片- A仪（皮尔斯）2小时。 </li><li>更换缓冲液和透析另外2小时在黑暗的房间温度。 </li><li>替换缓冲区和透析一夜之间在4 ° C黑暗环境中。 </li><li>商店纯化标记的聚合物，在-20 ° C。 </li></ol> 注意：在这项研究中，标准曲线是通过测量在670 nm的Cy5的NHS酯的排放强度构造。表征Cy5标记，标记的壳聚糖通过测量获得的排放标准曲线在670纳米的标签密度。吸光度也可能被用来确定标签的效率，但不执行。 C.量子点标记的DNA和Cy5聚合物的制备至QD的PDNA摩尔比保持在过剩（PDNA：QD≈1：2），以确保完整的量子点共轭PDNA。估计到每个质粒DNA标记的量子点的数量可以通过TEM成像或其他同等设施。在我们的研究中，每PDNA量子点的数量估计为1-3 TEM和单分子光谱。1使用Millipore公司的Milli - Q梯度准备在水（&gt; 18.0兆瓦，0.2um过滤） 。 <ol><li>计算所需的壳聚糖为10μgPDNA根据所需的N / P比，在壳聚糖溶液中的DNA溶液的负面磷酸盐的质子化胺的理论比例。 </li><li>添加到链霉亲和官能605QDs（Qdot 605 ITK的，Invitrogen公司，卡尔斯巴德，加利福尼亚州）的生物素化的PDNA解决方案。 </li><li>在室温孵育15分钟的黑暗的解决方案。 </li><li>量子点标记的DNA添加到50毫米硫酸钠溶液，使终体积200μL。 </li><li>稀释Cy5的壳聚糖，根据所需的N / P比Milli - Q水，使终体积200μL。 </li></ol> 注：请在黑暗的反应，以防止可能的漂白。 重要提示：谨慎使用Qdot 605 ITK™链霉亲和素共轭（ITK的系列），因为在这个目录中的量子点是设计为目的的FRET。定期Qdot系列共轭与PEG层，以防止非特异性结合，尤其是蜂窝标签。但是，这种额外的涂层扩大体 - 受体之间的距离，在Reduced的能量转移效率。 D.制造的SU - 8使用标准光刻大师<ol><li>硅片是食人鱼清理和烤在200℃5分钟。 </li><li>指定的主厚度为25μm的，旋涂硅晶片在2000转负型光刻胶（SU - 8 2025年，Microchem，牛顿，马），持续30秒。 </li><li>软烤的烤盘上斜了65晶圆° C /小时至95 ° C。 </li><li> 250mJ/cm 2暴露到紫外光灯（365nm）通过掩模薄膜含有微通道的设计（CAD /一流的服务，班登，或） 。 </li><li>暴露后斜了65烤盘上烤晶圆° C /小时至95 ° C。 </li><li>发展晶圆使用SU - 8光刻胶开发。 </li><li>图案的晶圆是很难一个烤盘烤坡道为65 ° C /小时至200 ° C。晶圆保持在200 ° C，至少5个小时，然后再逐步的晶圆冷却到室温。 </li></ol> 重要：逐渐升温过程中的SU - 8主烘烤过程中是必要的，否则的SU - 8结构可能从硅片或裂缝分离的SU - 8结构可能是由应力释放引起的。 E.副本成型PDMS的主人和粘接到玻璃盖<ol><li> SU - 8的主，是放置在一个称量船。 </li><li>混合有机硅弹性体和固化剂（聚二甲基硅氧烷，硅橡胶，Sylgard 184，道康宁，美联，心肌梗死）在10：1的比例。 </li><li>到SU - 8的主PDMS的混合物倒入离开称重船在真空干燥器中去除气泡。 </li><li>在65 ° C固化的PDMS为1-2小时。 </li><li>剥离泗母模的PDMS地带。 </li><li>打孔机通道的入口和流体设备的网点。 </li><li>清洁的PDMS地带用乙醇和玻璃盖，然后风干。 </li><li>治疗与氧等离子体清洗的PDMS地带和玻璃盖（1分钟20W）。 </li><li>立刻与玻璃盖债券的PDMS地带。 </li><li>留在烘箱在95℃过夜保税的微流体芯片。 </li></ol> 重要事项 ：等离子体处理和隔夜烘烤是必不可少的，以增强粘接强度。 F.显示器在微流体装置中形成的DNA Nanocomplexes <ol><li>水填充的微流体通道（，以确保有微流体通道内无气泡），在装货前的试剂，在实验过程中，以确保畅通。 </li><li>装入两个单独的玻璃注射器的量子点标记的DNA和Cy5标记的壳聚糖溶液，通过视频中所描述的油管。 </li><li>与微流体装置的两个进气口的连接管。要谨慎，不要在这个过程中引入任何空气。 20nL/min（PHD - 2000注射泵，Holliston，马）设置的流量，在层流条件下。 </li><li>检查在显微镜下的微。 </li><li>当流量稳定（约15至20分钟），QD介导FRET通道的中心应遵守。 </li><li>采取不同的位置沿通道的荧光照片（冷却CCD，Qimaging，加拿大不列颠哥伦比亚省）。 </li><li>分析与ImageJ和OriginLab的荧光图像。 </li></ol><img src="/files/ftp_upload/1432/1432fig1.jpg" alt="图1" /> 图1。 QD - FRET提供了一个敏感自组装的DNA Nanocomplexes发病迹象 <ol style="list-style-type:lower-alpha;"><li>量子点介导的荧光共振能量转移（QD - FRET）可以提供一个Polyplex公司稳定的定量和高度敏感的指示，在课外或细胞内环境允许发病的DNA和基因载体之间的相互作用明确检测。 FRET的一双，605QD和Cy5，被选择的基础上最大化供体和受体之间的光谱重叠和最大限度地减少潜在的串扰。这对福斯特距离69.4Å。 </li><li>自组装量子点的FRET的DNA nanocomplexes。 ，分别标有QD（能量捐助）和Cy5（能源受体），阴离子质粒DNA（PDNA）和阳离子基因载体。 QD - FRET nanocomplexes通过静电复凝聚形成。当激发波长为488，QD - FRET介导的Cy5的排放表示，形成一个紧凑和完整的纳米复合。可以计算的停留时间（T R）的措施，两条溪流从那里开会，正在调查的反应的立场，和平均血流速度（V）的距离（ X）。由于层流的性质，搅拌，只需要在接口的地方（每幅图像的中心）， 允许集体运输作为T R的函数的精确计算。时间分辨率，可以调整不同的applie三维流率。 （插图）的FRET -介导的信号，立即在两股气流相遇时的界面，表明具有约束力的是迅速的，发生在几毫秒内根据应用的流速。比例尺：100μm的。 </li></ol>

<ol>
	<li>Ho, Y. P., Chen, H. H., Leong, K. W., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+intracellular+stability+and+unpacking+of+DNA+nanocomplexes+by+quantum+dots-FRET.">Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.</a> J Control Release. 116, 83-89 (2006).</li><li>Chen, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+comparison+of+intracellular+unpacking+kinetics+of+polyplexes+by+a+model+constructed+from+quantum+dot-FRET.">Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET.</a> Mol. Ther. 16, 324-332 (2008).</li><li>Zhang, C. Y., Yeh, H. C., Kuroki, M., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Quantum-Dot-Based+DNA+Nanosensor.">Single-Quantum-Dot-Based DNA Nanosensor.</a> Nat Mat. 4, 826-831 (2005).</li></ol>

The authors have nothing to disclose.

<ul><li>我们的工作的意义： <ol><li>这是第一次尝试通过Q​​D - FRET的反应监测聚合物的DNA自组装纳米复合实时动力学（毫秒级的分辨率）在一个简单的微流体芯片。 </li><li> QD介导品提供了一个发病的分子间的相互作用和整个自组装过程中的高度敏感和定量的指示，而微流体提供了一个良好的控制微环境的空间分析在DNA纳米复合合成的过程。 </li><li>微流体芯片和纳米光子学集成提出了新的和有趣的方法，以调查任何类型的络合反应。 </li><li> QD - FRET的聚合物的DNA nanocomplexes可以很容易地建立结构与功能的关系的应用 1,2 </li></ol></li></ul>

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>SU-8</td>
<td>&nbsp;</td>
<td>MicroChem</td>
<td>SU-8 2025</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Plasmid DNA</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>pEGFP-C1</td>
<td>4.9 kb, MW = 3.3 x 106</td>
</tr>
<tr>
<td>LabelIT Biotin Labeling Kit</td>
<td>&nbsp;</td>
<td>Mirus Bio LLC</td>
<td>MIR 3400</td>
<td>Standard protocol yields labeling efficiency of approximately one label every 20-60 bp of double-stranded DNA, The density of labeling was adjusted in this work.</td>
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<tr>
<td>Streptavidin 605QD</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Qdot&reg; 605 ITK&trade; Streptavidin Conjugate</td>
<td>&nbsp;</td>
<tr>
<td>Cy5-NHS Ester</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>PA15101</td>
<td>&nbsp;</td>
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<td>Chitosan</td>
<td>&nbsp;</td>
<td>Vanson</td>
<td>390 kDa </td>
<td>83.5% deacetylated</td>
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<td>Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545C</td>
<td>No. 1; Size: 40 x 22mm</td>
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<td>Gastight Glass Syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>TLL series</td>
<td>50&mu;L to 500&mu;L depending on sample volume</td>
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<td>Tygon Tubing</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>0.02 ID, 100ft</td>
<td>Tygon Tubes Microbore, 0.02 ID, 100ft</td>
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<td>Connector</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>HTX-23R</td>
<td>Customized in length of 0.750"</td>
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<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD-2000</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Qimaging</td>
<td>Intensified Retiga Cooled</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Olympus</td>
<td>BX-51</td>
<td>100W mercury arc lamp</td>
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<td>ImageJ</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>v1.36b</td>
<td>http://rsb.info.nih.gov/ij</td>
</tr>
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<td>Origin Pro8</td>
<td>&nbsp;</td>
<td>OriginLab</td>
<td>Student Version</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>475AF40</td>
<td>Excitation filter in both channels</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595AF60</td>
<td>Emission filter in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>670DF40</td>
<td>Emission filter in QD-FRET channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>500 DRLP</td>
<td>Long pass dichroic in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595DRLP</td>
<td>Long pass dichroic in QD-FRET channel</td>
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combining qd-fret and microfluidics to monitor dna nanocomplex self-assembly in real-time

在基因组学的进展继续燃料的疗法，可以针对在细胞和分子水平发病机制的发展。通常在细胞内的功能，基于核酸疗法需要一个有效的细胞内传递系统。一个被广泛采用的方法是复杂的DNA与基因载体，通过静电自组装形成的nanocomplexes，促进细胞吸收DNA的同时保护对退化。挑战在于设计合理，高效的基因携带者，因为过早离解或过于稳定的约束力会损害细胞吸收和疗效。散装混合合成Nanocomplexes显示各种不同的细胞内拆包和贩运的行为，这是由于在规模和稳定nanocomplexes的异质性。这种异质性阻碍了自组装动力学的准确评估，并增加了其物理性质，化学性质相关的转染效率或生物活性的难度。我们提出了一个新颖的纳米光子学（即量子点的荧光共振能量转移）和微流体收敛特征下的自组装纳米复合层流的实时动力学。 QD - FRET发病的分子间的相互作用和整个合成过程中的定量测量提供了一个高度敏感的迹象，而微流体提供了一个良好控制的微环境，空间分析与高时间分辨率（毫秒）的过程。对于聚合物nanocomplexes的模型系统，在自组装过程中的两个不同的阶段被抓获这个分析平台。在微尺度获得的自组装过程中的动力学方面将特别适用于微反应器为基础的反应，这是许多微型和纳米尺度的应用有关的宝贵。此外，nanocomplexes可通过适当的设计microfludic设备，定制，和由此产生的QD - FRET的聚合物的DNA nanocomplexes可以随时建立结构与功能的关系的应用。

Watch this Scientific Journal Video about Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time at JoVE.com

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

我们提出了一个新颖和强大的集成纳米光子学（QD - FRET）技术和微流体调查，这是预计未来基于核酸疗法提供更好的控制和统一的和可定制polyplexes的合成聚电解质polyplexes形成。

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

combining-qd-fret-microfluidics-to-monitor-dna-nanocomplex-self

Research

JoVE Journal

Biology

11.0K 次观看.  Johns Hopkins University. 我们提出了一个新颖和强大的集成纳米光子学（QD - FRET）技术和微流体调查，这是预计未来基于核酸疗法提供更好的控制和统一的和可定制polyplexes的合成聚电解质polyplexes形成。

视频: Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

海洋微生物的趋化反应的微观尺度的营养层

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

科研

生物学

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

使用微流体的细菌的趋化作用的研究 - 访

Applying Microfluidics to Electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice.  By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice.  This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate.  This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate.  These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants.  This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

应用微流体电

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

数字微流体进行自动蛋白质组学处理

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

通过流动细胞测定检测和描述 Vivo 中的蛋白质自组装

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

阿拉伯多普西斯局部和系统伤口信号的广域实时成像

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装