We thank Vladimir Polejaev (Yale West Campus Imaging Core) for the design and construction of the polarized TIRF microscope, David Baddeley (Yale University) for help with two-color detection instrumentation, and James E. Rothman (Yale University) and Ben O'Shaughnessy (Columbia University) and members of their groups for stimulating discussions. EK is supported by a Kavli Neuroscience Scholar Award from the Kavli Foundation and NIH grant 1R01GM108954.

作者宣称，他们没有竞争的经济利益。

成功地实施这里所描述的SUV-SBL融合法的关键取决于几个关键步骤，例如蛋白质的功能性重组到脂质体中，获得良好的质量SBLs，并选择合适的摄像参数，以检测单个分子。虽然这可能需要一些时间和努力才能成功，一旦试验成功实施，它提供了无法从简介中讨论的任何其他体外实验融合了丰富的有关融合过程的信息。率在该越野车停靠到和保险丝与SBL，对接至融合时间的分布，脂类扩散率?...

膜融合为脂质和蛋白质，分泌，施肥，发展的细胞内运输所需的通用生物过程，和包膜病毒进入宿主生物体1-3。对于大多数细胞内聚变反应包括通过胞吐的激素和神经递质的释放，融合2脂质双层的能量由形成同源可溶性N-乙基马来酰亚胺-敏感因子附着蛋白受体 （SNARE）的蛋白质之间的四螺旋束的提供，锚定在囊泡（v-SNARE）和分别的靶膜（叔SNARE）4，。突触小泡胞吐作用是最严格调控的聚变反应和动作电位1,4,5到货后一毫秒内发生。的融合孔，两个熔断隔间之间的初始连接，可以闪烁打开和再密封或不可逆5-7扩大之前关闭多次。前者结果在短暂的"亲吻与运行"的融合，而后者导致完全融合。理事融合这两种模式和调节孔径闪烁机制之间的平衡因素不能很好地理解5,8。 SNARE蛋白所需的胞吐作用;突触囊泡融合是由神经毒素9时SNARE蛋白裂解取消。使用小单层囊泡（SUV的）堆积聚变实验表明，斯耐尔不仅必需的，但也足以驱动膜融合10。在此批量测定中，重构用v-SNARE蛋白（V-SUV）的SUV被掺杂有荧光磷脂（N - （7-硝基2-1,3-苯并恶二唑-4-基）-phosphoethanolamine（NBD-PE）和（ N - （丽丝胺罗丹明B磺酰基）-phosphoethanolamine（LR-PE）和含叔SNARE蛋白（T-SUV）最初的v型的SUV NBD-PE的荧光被荧光共振能量转移淬无标签的小泡混合（FRET ），以LR-PE作为实验室ELED v型越野车与未标记叔SUV的融合，在现在结合膜的荧光面密度降低，在NBD-PE的荧光所产生的增加报告脂质混合10的程度。象散法是容易设置和分析，它已被广泛用来研究SNARE介导的融合10-14的机制。然而，它有一些限制，如低灵敏度和时间分辨率差。最重要的是，作为一个集合进行测定，平均结果​​在使半融合中间体难以检测对接和融合区分，以及所有事件。 在过去的十年几组，包括我们，已经开发了新的分析方法来监控在单级囊泡融合15-27事件。厦和他的同事用拴在表面上的V-SUV和监控他们与免费的T SUV的融合18,19。脂质混合是一对脂相连的荧光团之间的FRET使用EM监视层状分别在v型和叔越野车，使用全内反射荧光（TIRF）显微镜18。后来，Brunger的实验室使用单一脂质标签的物种连同内容标记物的脂质和内容混合20,28同时检测。既脂质和内容标记被包括在高，自猝灭浓度;融合SUV的未标记导致荧光脱猝灭20,28。 其他人已经融合v型越野车与T-SNARE蛋白15-17,21-27,29重组平面双层。目标（含T-SNARE）双层更好地模拟与一台质膜小，高度弯曲的囊泡的生理融合过程中的平面形状。该斯泰纳姆组采用与T-SNARE蛋白重组孔跨越膜，悬浮在多孔氮化硅基板，并检测使用激光共聚焦显微镜23个人的V型SUV的融合。其他˚F使用V型越野车与T-SNARE蛋白重组平面双层，支持在玻璃基板上15-17,21,22,24-27,29。使用支持的双层（SBLs）的巨大优点在于，TIRF显微镜可以用于检测具有优良信噪比并且在不脱离自由V形的SUV干扰对接和融合事件，虽然使用微流体使用还提供了单事件分辨率标准的远场荧光显微镜24。 一个主要问题是底物的相互作用双层是否以及如何影响支持双层质量和融合的过程。早期工作取得使用该直接支持的玻璃或石英衬底上15-17平面SBLs的。这些SBLs被吸附制成，爆裂，扩散和在衬底上叔SUV膜融合。它很快实现，然而，省略了密钥t-SNARE组分，SNAP25，从以这种方式制备SBLs导致V形的SUV对接和融合动力学indistinguishable使用完整的T-17 SNARE蛋白获得的。因为SNAP25 在体内 30,31绝对必需的融合，这些早期的尝试生理相关投入的问题。塔姆的研究小组通过使用更好的控制支持双层形成21战胜了这一挑战。它使用朗缪尔-布洛杰特沉积为SBL的无蛋白质的第一小叶，接着用叔-越野车21，其单层融合。这导致SNAP25依赖性融合。 以避免与直接支撑在玻璃基板上，而不需要使用朗缪尔-布洛杰特法的双层相关的潜在假象，Karatekin 等人介绍了柔软，水合聚（乙二醇）（PEG）的双层和基板24之间的缓冲。该变形也导致SNAP25依赖性融合24。垫着柔软的聚合物层上双层一直被称为更好地保存跨膜蛋白质的流动性和功能32，并与病毒融合33研究中使用。此外，聚乙二醇化双层似乎保留一定的能力进行自我修复，而且非常坚固34,35。首先，商购，脂质连接PEG链的一小部分被包括在叔SUV膜。当这些叔SUV的突发，并形成在玻璃基板上的平面双层，一个PEG刷覆盖平面双层的两个小叶。因为平面双层形成通过叔SUV的周围到所述亲水玻璃表面的PEG链的粘附驱动，脂质体破裂和平面双层形成相对不敏感，以所使用的脂质组合物。然而，当包括大量的胆固醇的，增加了越野车的粘结性时，越野车可以不自发破裂。如果是这种情况，可以采用渗透休克或二价离子，以帮助平面双层形成25。 如在本AP如上所述，proach的PEG刷覆盖所述平面的两侧，支持双层。面对微流体流动通道的刷有助于防止其也通常覆盖有聚乙二醇层传入v型越野车的非特异性粘附。 v型和叔SNARE复合体的形成从膜远侧N-末端并且前进朝向膜近端域36阶段开始。为V形的SUV与叔SBL中，v型和叔SNARE N-末端需要突出的PEG刷，这似乎是在测定条件下，上述情况下进行交互。刷高度可适于通过改变PEG化脂质的密度和PEG链长度37,38学习比斯耐尔其它蛋白质。所述PEG刷子覆盖定影双层的近侧表面中的另一个好处是，它们模仿它们填充有每平方微米39 30,000-40,000整合膜蛋白生物膜的拥挤的环境。就像在此测定的PEG链，所述REPU覆盖生物膜lsive蛋白层需要被推开以允许两个磷脂双层之间的接触为发生融合。 微流体流动通道在该测定中使用的，因为它们提供独特的优点。首先，微流体流使T-的SUV更均匀的沉积扩散和融合形成了T-SBL。其次，小声道音量（&lt;1微升）减少样品消耗。第三，所需的小体积允许下恒定流量进行的整个实验。流动弱除去，大概非特异性，从​​SBL 16附着v型越野车。它还维护V型的SUV的T-SBL以上，简化动力学分析17的密度不变。最后，停靠囊泡容易从由流25进行自由的区分。第四，多个微流体通道可以在相同的盖玻片一起使用，每个探测不同的条件。这使得杜里条件比较纳克相同的实验运行。类似的方法已被用于面包车Oijen组研究流感病毒和缓冲SBLs 33之间的融合。 在TIRF显微镜，渐逝场的指数衰减（以衰减常数〜100nm）的界限荧光激发到那些在玻璃 - 缓冲界面的非常接近的分子。这最小化是更远离荧光分子的贡献，增加信噪比，并且允许用10-40毫秒的帧的曝光时间单分子灵敏度。渐逝场也导致在融合的信号增加:从SUV标记的脂质转移到SBL，他们发现自己，平均而言，在更强的激励场。这增加了荧光较大的脂质体更强。 如果偏振光是用来产生渐逝场，附加效果有助于对T中的荧光变化从SUV到SBL标签的转让（BOT）。某些脂质染料具有与相对于它们所嵌入的双层的优选平均角度定向的跃迁偶极。这将在通过荧光团时，他们在SUV 兑 SBL发射的荧光的量的差异，由于偏振光分束将在两个膜激发染料不同。对于前者，激发光束将与周围的球形的SUV取向过渡偶极子相互作用，而对于后者，偶极取向将由扁平SBL几何局限。例如，当S偏振入射的光（偏振光垂直于入射平面）时，激发是更有效率的，当染料在SBL比在SUV脂质染料跃迁偶极方向平行于膜29,40一（如直接投资收益还是没41-43）。掺杂了这样的荧光团SUV似乎暗淡时，码头上SBL（ 图7，Representat<html>香港专业教育学院结果）。作为融合孔打开，并连接在SUV和SBL膜，荧光探针扩散到SBL和变得更容易被s偏振光渐逝场25,27,29激发。因此，周围的融合位点整合的荧光信号从所述的SUV染料转移到SBL 27（ 图3和图7）期间急剧增加。有助于信号伴随融合脱猝灭的荧光标记的时转移到SBL因为它们稀释变化的另外一个因素。相比，在这里描述的测定消逝场衰减和极化效应脱猝灭的贡献通常是次要的，因为只有一小部分（ <img alt="式（1）" src="/files/ftp_upload/54349/54349eq1.jpg" /> ）脂质的标记。 在融合信号增加可以利用通过比较时间来推断融合孔性质，1"SRC ="/文件/ ftp_upload / 54349 / 54349eq2.jpg"/&gt;，对于脂质通过的孔是自由可渗透脂质实际释放时间逃逸要求<img alt="式（1）" src="/files/ftp_upload/54349/54349eq3.jpg" /> 。如果两个时间尺度是可比，将它的结论是，孔呈现给脂质流动阻力小。然而，如果实际的释放时间是显著比时间为扩散限制释放更长的时间，这将表明一个过程中，如孔隙抖动，延迟脂质释放。扩散限制释放时间， <img alt="式（1）" src="/files/ftp_upload/54349/54349eq2.jpg" />取决于定影脂质体和脂质扩散的大小;其估计需要这两个参数来量化。测定的单分子灵敏度允许脂质扩散到其释放到SBL为每个聚变事件26之后追踪几个单脂的荧光团测定。每一熔断囊泡的大小可通过将（i）的单脂质染料的强度，（ii）在所有荧光团在融合转移到SBL中围绕一个停靠站点的总荧光变化，（iii）该已知标记的SUV的密度来估计27脂质，和（iv）每脂质的区域。对于许多融合事件中，发现了实际脂质释放时间要慢得多比由扩散控制的释放27预期，如先前指出的假定均匀的SUV大小44。假设脂质释放迟缓是由于毛孔闪烁，定量模型允许"毛孔开放"的估计，时间的部分孔隙依然融合27期间开放。 每当实际，以测试同时使用脂质和可溶性内容标签融合的机制是很重要的。例如，脂质释放可以通过比孔隙闪烁等工艺，如由SNARE蛋白是环绕吨脂质扩散的限制被延迟他孔隙。如果是这样的话，然后松开的内容将先于脂质标签释放，所提供的孔径是大到足以允许可溶性探针通道。在方法的一个更基本的缺陷可能是通过一个狭窄的融合孔的SBL连接到囊泡已经基本上保留了其融合前形状发生标记的脂质转移到SBL的假设。脂质转移到SBL也可以从融合孔与随之而来的，超极本的SUV进入SBL膜的迅速崩溃的快速散瞳造成的，正如上面所提到仅仅基于29脂质发布的数据。同时监测血脂和内容同时发布，发现许多气孔释放他们所有的脂质标签后重新密封，但保留了他们的一些可溶性的货物27。这表明，至少一些脂质体不塌陷到SBL融合之后，并且该脂质染料转移到SBL通过融合孔发生。此外，LIPID和内容，同时发布27发生，使得它不可能脂质释放迟缓，是由于受到周围孔45 SNARE蛋白阻碍脂质扩散的。 未监测溶于内容发布一款SUV-SBL融合协议之前由Karatekin和罗斯曼25公布。在这里，最近的事态发展都包括在内，脂质即同步监测和内容发布和SUV，脂质，并融合孔特性27的推定。该协议开始于制备微流体细胞，通过结合的聚（二甲基硅氧烷）制成（PDMS）弹性体包含块槽用玻璃盖玻片25的指令。接着，制备V-SUV的与两个脂质和内容标记进行说明。第4和5用于组装微流体细胞， 原位形成的SBLs并检查缺陷和流动性，引入VS的提供指令UVS到流动细胞和融合事件的检测。部分6提供用于数据分析的指令。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q (MQ) water</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>J.T. Baker</td>
			<td>3040-05</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 190 Proof</td>
			<td>Decon</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Chemical</td>
			<td>A416P4</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td rowspan="3">AmericanBio</td>
			<td>AB00892</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cholride (KCl)</td>
			<td>AB01915</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>AB00490</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-[2-hydroxyethyl] piperazine-N&#39;-[2-ethanesulfonic acid] (HEPES)</td>
			<td>AmericanBio</td>
			<td>AB00892</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros Organics</td>
			<td>409911000</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffers</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES-KOH buffer (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>25 mM HEPES-KOH, 140 mM KCl, 100 &#956;M EGTA, 1 mM DTT</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solvents</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform&#160;</td>
			<td>J.T. Baker</td>
			<td>9180-01</td>
			<td>in glass bottle, CAUTION, wear PPE</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>J.T. Baker</td>
			<td>9070-03</td>
			<td>in glass bottle, CAUTION, wear PPE</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liposome preparation&#160;</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gastight Hamilton syringe</td>
			<td>Hamilton</td>
			<td>var. sizes</td>
			<td>only use glass sringe with solents (Chlorophorm/ Methanon, 2:1, v/v)</td>
			<td><a href="http://www.hamiltoncompany.com/">http://www.hamiltoncompany.com</a></td>
		</tr>
		<tr>
			<td>Glass tubes Pyrex Vista 11 ml, 16x100 mm screw cap culture tube</td>
			<td>Pyrex&#160;</td>
			<td>70825-16</td>
			<td>clean thoroughly, rinse with chloroform</td>
			<td><a href="http://catalog2.corning.com/LifeSciences/">http://catalog2.corning.com/LifeSciences/</a></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 16:0-18:1 PC (POPC)</td>
			<td rowspan="7">Avanti Polar Lipids</td>
			<td>850457</td>
			<td rowspan="7">Lipids come dissolved in CHCl3 or as lyphilized powder in sealed vials. Aliquot upon opening. Store extra as dried lipid films under inert atmosphere at -20 &#176;C. Keep stocks in CHCl3/MeOH (2:1, v/v) at -20 &#176;C. let come to RT before opening</td>
			<td rowspan="7"><a href="http://www.avantilipids.com/">http://www.avantilipids.com/</a></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt), 18:1 PS (DOPS)</td>
			<td>840035</td>
		</tr>
		<tr>
			<td>1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine, 18:0-20:4 PE (SAPE)</td>
			<td>850804</td>
		</tr>
		<tr>
			<td>L-&#945;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt), Brain PI(4,5)P2</td>
			<td>840046</td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), 18:1 NBD PE</td>
			<td>810145</td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 18:1 PEG2000 PE</td>
			<td>880130</td>
		</tr>
		<tr>
			<td>cholesterol (ovine wool, &#62;98%)</td>
			<td>700000</td>
		</tr>
		<tr>
			<td>DiD&#39; oil; DiIC18(5) oil (1,1&#39;-Dioctadecyl-3,3,3&#39;,3&#39;-Tetramethylindodicarbocyanine Perchlorate)</td>
			<td>Molecular Probes</td>
			<td>D-307</td>
			<td></td>
			<td><a href="https://www.thermofisher.com/">https://www.thermofisher.com/&#160;</a></td>
		</tr>
		<tr>
			<td>Rotavapor R-210</td>
			<td>Buchi</td>
			<td>R-210</td>
			<td>heat bath above Tm of lipids used</td>
			<td><a href="http://www.buchi.com/">http://www.buchi.com/</a></td>
		</tr>
		<tr>
			<td>OG n-Octyl-&#946;-D-Glucopyranoside</td>
			<td>Affymetrix</td>
			<td>0311</td>
			<td>store at -20&#176;C, let come to RT before opening</td>
			<td><a href="https://www.anatrace.com/">https://www.anatrace.com/</a></td>
		</tr>
		<tr>
			<td>Shaker - Eppendorf Thermomixer R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
			<td><a href="https://www.eppendorf.com/">https://www.eppendorf.com/</a></td>
		</tr>
		<tr>
			<td>Slide-A-Lyze Dialysis Cassettes, 20K MWCO, 3 mL</td>
			<td>life technologies</td>
			<td>66003</td>
			<td></td>
			<td><a href="https://www.lifetechnologies.com/">https://www.lifetechnologies.com/</a></td>
		</tr>
		<tr>
			<td>Bio-Beads SM-2 Adsorbents</td>
			<td>Bio-Rad</td>
			<td>1523920</td>
			<td></td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
		</tr>
		<tr>
			<td>OptiPrep&#160;Density Gradient Medium</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr>
			<td>Ultracentrifugation tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm&#160;</td>
			<td>Beckman Coulter</td>
			<td>41121703</td>
			<td></td>
			<td><a href="https://www.beckmancoulter.com/">https://www.beckmancoulter.com/</a></td>
		</tr>
		<tr>
			<td>Beckman SW41 Ti rotor</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SuflorhodamineB</td>
			<td>Molecular Probes</td>
			<td>S-1307</td>
			<td></td>
			<td><a href="https://www.thermofisher.com/">https://www.thermofisher.com/&#160;</a></td>
		</tr>
		<tr>
			<td>Econo-Column&#160;Chromatography Columns, 2.5 &#215; 10 cm</td>
			<td>Bio-Rad</td>
			<td>7372512</td>
			<td></td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
		</tr>
		<tr>
			<td>Sepharose CL-4B</td>
			<td>GE Healthcare</td>
			<td>17-0150-01</td>
			<td></td>
			<td><a href="http://www.gelifesciences.com/">http://www.gelifesciences.com/</a></td>
		</tr>
		<tr>
			<td>SYPRO Orange Protein Gel Stain</td>
			<td>Molecular Probes</td>
			<td>S-6650</td>
			<td>5,000X Concentrate in DMSO</td>
			<td><a href="https://www.lifetechnologies.com/">https://www.lifetechnologies.com/</a></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS block</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone elastomer kit, PDMS</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td></td>
			<td><a href="http://www.dowcorning.com/">http://www.dowcorning.com/</a></td>
		</tr>
		<tr>
			<td>Pyrex glass petri dish, 150 x 20 mm, complete with cover</td>
			<td>Corning</td>
			<td>3160-152</td>
			<td></td>
			<td><a href="http://catalog2.corning.com/LifeSciences/">http://catalog2.corning.com/LifeSciences/</a></td>
		</tr>
		<tr>
			<td>Hole puncher - Reusable Biopsy Punch, 0.75mm</td>
			<td>World Precision Instruments</td>
			<td>504529</td>
			<td></td>
			<td><a href="http://www.wpi-europe.com/">http://www.wpi-europe.com/</a></td>
		</tr>
		<tr>
			<td>Manual Hole Punching Machine&#160;</td>
			<td rowspan="2">SYNEO</td>
			<td>MHPM-UNV</td>
			<td></td>
			<td rowspan="2">http://www.syneoco.com/</td>
		</tr>
		<tr>
			<td>Drill .035 x .026 x 1.5 304 SS TiN coated round punch</td>
			<td>CR0350265N20R4</td>
			<td>drill diameter: 0.9 mm</td>
		</tr>
		<tr>
			<td>Tygon Microbore tubing, 0.25 mm ID, 0.76 mm OD</td>
			<td rowspan="2">Cole-Parmer</td>
			<td>06419-00</td>
			<td>0.010&#34; ID, 0.030&#34; OD</td>
			<td rowspan="2"><a href="http://www.coleparmer.com/">http://www.coleparmer.com/</a></td>
		</tr>
		<tr>
			<td>Silicone Tubing (0.51 mm ID, 2.1 mm OD</td>
			<td>95802-00</td>
			<td>0.020&#34; ID, 0.083&#34; OD</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass -&#160; cleanroom cleaned</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Schott Nexterion cover slip glass D</td>
			<td>Schott</td>
			<td>1472305</td>
			<td></td>
			<td><a href="http://www.us.schott.com/">http://www.us.schott.com/</a></td>
		</tr>
		<tr>
			<td>plasma cleaner</td>
			<td>Harrick</td>
			<td>PDC-32G</td>
			<td></td>
			<td><a href="http://harrickplasma.com/">http://harrickplasma.com/</a></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pTIRF setup and accessories</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IX81 microscope body</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
			<td><a href="http://www.olympus-lifescience.com/en/">http://www.olympus-lifescience.com/en/</a></td>
		</tr>
		<tr>
			<td>EM CCD camera</td>
			<td>Andor</td>
			<td>ixon-ultra-897</td>
			<td></td>
			<td><a href="http://www.andor.com/">http://www.andor.com/</a></td>
		</tr>
		<tr>
			<td>Thermo Plate, heated microscope stage</td>
			<td>Tokai Hit</td>
			<td>MATS-U52RA26</td>
			<td></td>
			<td><a href="http://www.tokaihit.com/">http://www.tokaihit.com/</a></td>
		</tr>
		<tr>
			<td>1 ml hamilton glass syringes (4x)</td>
			<td>Hamilton</td>
			<td>81365</td>
			<td></td>
			<td><a href="http://www.hamiltoncompany.com/">http://www.hamiltoncompany.com</a></td>
		</tr>
		<tr>
			<td>syringe pump</td>
			<td>kd Scientific</td>
			<td>KDS-230</td>
			<td></td>
			<td><a href="http://www.kdscientific.com/">http://www.kdscientific.com/</a></td>
		</tr>
	</tbody>
</table>

snare-mediated fusion of single proteoliposomes with tethered supported bilayers in a microfluidic flow cell monitored by polarized tirf microscopy

在膜融合的泛在处理的融合孔的开口在两台以前分开的隔室之间的第一个连接。在通过胞外分泌神经递质或激素释放的融合孔可以瞬时打开和关闭多次，规范货物释放动力学。孔动力学也确定囊泡循环的模式;在短暂的"吻了就跑"融合不可逆转再加盖印章的结果，而扩张，从而达到完全融合。为了更好地了解哪些因素支配孔隙动力学，我们开发了一种测定中使用偏振全内反射荧光（TIRF）显微镜用单分子灵敏度和〜15毫秒的时间分辨率在体外系统中的生物化学良好定义并监控膜融合。融合的荧光标记的含V-SNARE蛋白（V-越野车）与平面双层轴承T-SNARE蛋白小单层囊泡，支持软聚合物垫（T-SBL，T-支持双层），被监视。该法采用微流体流动通道，以确保最少的样品消耗，同时提供越野车的密度不变。利用在熔化过程中从SUV脂质标签转移到SBL的快速信号增强，脂质染料转移的动力学进行监测。 TIRF显微镜的灵敏度可跟踪单个荧光脂质标签，从中脂质扩散和SUV的大小可以推断为每个融合事件。脂质染料释放时间可以更长比预期通过永久开放的毛孔畅通的通道。使用该假设脂质释放的相位差是由于孔隙闪烁，细孔"开放"，该时间孔隙保持熔融时打开的分数的模型，可以进行估计。可溶性标记可以在SUV的脂质和可溶性货物放行的同步监测进行封装。这种测量表明失去可溶性货物的一小部分经过一些孔隙可以重新密封。

SBL质量 这是至关重要的事先验证SBL的质量和流动性，以融合实验。在一个微流体通道的底部，玻璃侧的荧光应均匀，而没有任何明显的缺陷。如果气泡虽然经过通道，它通常留下的SBL明显的疤痕。如果有如此大规模的疤痕/缺陷，不要使用该通道。有时越野车可能附着到衬底上，但未能突发和形成连续SBL。如果是?...

Watch this Scientific Journal Video about SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy at JoVE.co

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

在这里，我们提出了一个协议来检测使用偏振TIRFM脂质体和微流体通道支持双层之间的单，SNARE介导的融合事件，与单分子敏感性和〜15毫秒的时间分辨率。脂质和可溶性货物放行可同时检测。脂质体大小，脂质扩散率，和融合孔性质被测量。

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

snare-mediated-fusion-single-proteoliposomes-with-tethered-supported

Research

JoVE Journal

Neuroscience

10.8K 次观看.  Yale University School of Medicine. 在这里，我们提出了一个协议来检测使用偏振TIRFM脂质体和微流体通道支持双层之间的单，SNARE介导的融合事件，与单分子敏感性和〜15毫秒的时间分辨率。脂质和可溶性货物放行可同时检测。脂质体大小，脂质扩散率，和融合孔性质被测量。 

视频: SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

单细胞的多巴胺释放，同时电压钳和电流分析法测量

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

科研

神经科学

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

神经元共培养与单细胞精度的制备

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

A Simple Flight Mill for the Study of Tethered Flight in Insects

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have shown that flight mills are valuable tools for the experimental study of insect flight behavior, allowing researchers to examine how factors such as age, host plants, or population source can influence an insects' propensity to disperse. Flight mills allow researchers to measure components of flight such as speed and distance flown. Lack of detailed information about how to build such a device can make their construction appear to be prohibitively complex. We present a simple and relatively inexpensive flight mill for the study of tethered flight in insects. Experimental insects can be tethered with non-toxic adhesives and revolve around an axis by means of a very low friction magnetic bearing. The mill is designed for the study of flight in controlled conditions as it can be used inside an incubator or environmental chamber. The strongest points are the very simple electronic circuitry, the design that allows sixteen insects to fly simultaneously allowing the collection and analysis of a large number of samples in a short time and the potential to use the device in a very limited workspace. This design is extremely flexible, and we have adjusted the mill to accommodate different species of insects of various sizes.

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

一个简单的飞行磨的系留飞行的昆虫研究

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 &#181;m in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.

Here, we present a protocol for the formation of lipid bilayers using a contact bubble bilayer method. A water bubble is blown into an organic solvent, whereby a monolayer is formed at the water-oil interface. Two pipettes are manipulated to dock the bubbles to form a bilayer.

用于补丁夹紧器的接触气泡层的脂质双层实验

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...

Three-dimensional Navigation-guided, Prone, Single-position, Lateral Lumbar Interbody Fusion Technique

Lateral interbody fusion provides a significant biomechanical advantage over the traditional transforaminal lumbar interbody fusion due to the large implant size and optimal implant position. However, current methods for lateral interbody cage placement require either a two-staged procedure or a single lateral decubitus position that precludes surgeons from having either full access to the posterior spine for direct decompression or comfortable pedicle screw placement.
Herein is one institution's experience with 10 cases of a prone single-position approach for simultaneous access to the anterior and posterior lumbar spine. This allows both lateral lumbar interbody cage placement, direct posterior decompression, and pedicle screw placement, all in one position. Three-dimensional (3D) navigation is utilized for increased precision in both approaching the lateral spine and interbody cage placement. The traditional blind psoas muscle tubular dilation was also modified. Tubular retractors and lateral vertebral body retractor pins were used to minimize the risks to the lumbar plexus.

The single-position, prone, lateral approach allows for both lateral lumbar interbody placement and direct posterior decompression with pedicle screw placement in one position.

三维导航引导，俯卧，单位，侧腰椎椎体间融合技术

Lateral interbody fusion provides a significant biomechanical advantage over the traditional transforaminal lumbar interbody fusion due to the large ...

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

在微流体装置中生成具有功能性神经肌肉接头的人类运动单元

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...