我们想感谢DA库尔特（CHOP）的，允许我们使用他的双光子显微镜双光子图像显示在视频开始，和H.高野收购与我们分享他的技术专长。

第1部分：组织编制<ol><li>连续剥离150 - 200G（2-3周龄）哈特利几内亚，已经由吸入异氟醚和断头麻醉的猪小肠黏膜下和肌间神经丛制剂隔离。安乐死后，小肠切除和温暖，含氧的解决方案冲洗管腔，其内容删除。我们使用中等199辅以HEPES 25毫米，5毫米碳酸氢钠3和2毫米谷氨酰胺，和pH值调整到7.4（提到，从现在开始，M199 - DM），已在37预热° C。然而，该协议的所有剩余的步骤，将开展在室温下。 </li><li>肠道段，长8-10厘米，被转移到一个Sylgard菜含有M199 - DM，沿肠系膜边界打开，并安装黏膜侧使用昆虫引脚。这是非常重要的是保持张力均匀，使组织后穿针展品定期对齐的粘膜绒毛行长方形。 </li><li>使用直提示优良杜蒙钳，在几乎水平的位置，它很容易抓取的绒毛，在一端的准备和甩开沿纵轴组织剥离的黏膜。当样品是均匀寄托的，粘膜的丝带可以在少数个别笔划分开，露出的粘膜下层，包含粘膜下神经节和粘膜下血管。 </li><li>对于罚款的丛清扫，我们建议使用在解剖显微镜，暗场照明。事实上，这种类型的照明所造成的伪三维效果，有助于区分个别层，从而防止或尽量减少仪器造成损害。 </li><li>为了使粘膜下准备，我们按照赫斯特和McKirdy 4经典的程序，要求：a）作出削减在粘膜下层环形肌纤维的方向，可以做到这一点，使用非常细的剪刀（如莫里亚MC19或解除其当量）;二）粘膜下层，轻轻地用细镊子，而压低使用罚款剪刀圆形肌肉层; C）沿纵向边缘切割，分离过程，以摆脱其余的粘膜下层预习; D）第二个切下面的环形肌纤维，在距离第一个定义粘膜下准备的最终大小方向。导致粘膜下段，这是只有30至50微米厚，并有一个游丝外观，可转移到另一个Sylgard菜含有新鲜M199 - DM，轻轻地寄托和无张力，并保存供日后使用含氧室。 </li><li>原来肠道段，现在被剥夺黏膜和黏膜下层，环形肌暴露出来，仍然可以用于产生肌间神经丛准备。要揭露肌间神经丛，必须尽可能的圆形肌肉层可能消除。这可以通过使用提示在前面的步骤就业同一条直线罚款钳。纤维束可以拉轻轻地走，首先从一个，然后从另一端的准备。裸露的肌间神经丛不能从纵向的肌肉和底层浆膜层分开。因此，什么是所谓的肌间神经丛准备沿引脚，可切段。由于它含有活的肌肉，它是可取的，它非常松散，针中含有含氧M199 - DM菜，直到生理实验开始。 </li><li>由于黏膜是主要的ENS的传感器，和所有的效应也嵌入在肠壁，是可能的解剖半完整的准备工作，维护整个反射通路。这些定制设计的准备工作需要在1.3中描述的步骤的组合。到1.6，适用于离体肠管段的邻近地区。 </li><li>几分钟之前，实际的光学实验，准备选择一个安装在试验室，确保张力均匀分布和组织就在于对Sylgard底部完全平坦。这是取得了非常精细，可以各地外围没有造成组织损伤定位与多个引脚的标本。 </li></ol>第2部分：光学记录的组织样本的制备<ol><li> （可选）为了防止背景荧光染料结合残留的平滑肌和结缔组织，可孵育30-60分钟M199 - DM含胶原酶VII（≤50 U /毫升）和蛋白酶九（≤0.5微克两个孤立的丛/毫升）。酶处理的筹备工作，应清洗，并保持一夜之间，M199 - DM加上10％的动物血清（如马或牛）和抗生素（青霉素，100U/ml链霉素，100微克/毫升），在商会饱和无线网络日95％O 2和5％的CO 2。这个过程中，我们有多年的使用，因为它明显增强信号信噪比的光录音，不得对初学者很有帮助，应谨慎采用。事实上，在夹层中损坏了准备运行在酶处理瓦解的危险。然而，即使在酶处理的情况下，我们建议的筹备工作保持在隔夜M199 - DM，与血清和抗生素。这一步促进急性夹层动脉瘤后损伤组织的愈合，并增加了样品的透明度。 </li><li>不能实现电活动的光学记录在筹备中的肌肉收缩。因此，要防止肌肉收缩时，肌间神经丛，几分钟录制之前我们添加2微米硝苯地平M199 - DM洗澡组织的光学实验。 </li></ol>第3部分：染色与DI - 4 - ANEPPDHQ <ol><li> DI - 4 - ANEPPDHQ商业冻干形式提供。我们建议使用纯乙醇溶解，使一个集中的股票解决方案[如15-20毫克/毫升。这个股票，保存在-20 ° C时，是非常稳定。事实上，在我们的实验室，我们刚刚结束的这个原因，我们取得了近6年前的染料股票解决方案的最后一滴。 </li><li>染色液应准备在M199 - DM，如果该协议要求，应包含2微米硝苯地平。对染料的最终浓度可高达5微克/毫升和不应高于50微克/毫升。然而，这取决于该股票的浓度，将一定量的乙醇染色液中，应考虑。例如，染色溶液与50μg/ ml的染料将包含〜0.3％的乙醇，如果从15毫克/毫升的股票，但如果股票只有5毫克/毫升的浓度接近1％的乙醇。 </li><li>染色，准备被淹没在染料溶液10-30分钟。在这一时期结束后，染料被冲走，替换由新鲜，含氧M199 - DM。由于DI - 4 - ANEPPDHQ是一种膜impermeant染料，荧光只有在脂质环境，健康的神经元从一个彩色的肠道准备应具备的外观空气球。 </li></ol>第4部分：电刺激<ol><li>刺激可通过电压或电流驱动。我们用电流刺激，计算机控制，4通道的刺激发生器的最大输出每通道16毫安发表。 </li><li>在一个给定的实验中使用的电极模型是由实验的目标。可用于细胞内吸液管或修补电极时，我们的目标是一个特定的神经元的刺激。当刺激的目的是激活在一个给定的节的一个神经元以上，外电极接触结缔组织均可使用。研究互连节，大，低电阻电极和/或可以采用多元素的电极阵列，但是，我们一般使用电流刺激时，多元素的数组成了唯一的选择。许多型号的外电极，以及一些线性电极阵列，都是现成的，从商业来源。 </li><li>电极可使用不同类型micromanipulators的定位。为了避免由长而细桶引起的振动，我们赞成使用相邻的试验室，可定位的磁基地小micromanipulators，并支持灵活的钨电极与瘦，但很死板的金属条桶。 </li></ol>第5部分：灌注我们使用一个通道输入含氧的解决方案，和其他的输出，2通道蠕动泵。为了消除灌注引起的机械干扰，泵，光学数据采集期间关闭的。 第6部分：多个站点的光学记录膜电位器<ol><li>光学元件： 多个站点的光学记录膜电位的仪器，包括一个NeuroCCD - SM相机（RedShirtImaging）和1:10 demagnifier /中继镜头，装在一个堂堂正正的显微镜三目镜筒。显微镜移动的XY转换，独立的一个固定的主动隔振表顶部的阶段。为了减少空气中的振动，整个装置所包围的沉重的声学窗帘和聚酯薄膜屋顶。 EPI -照明是提供一个100W的卤钨灯超低纹波反馈稳压电源供电的灯。入射光是准单色，采用热过滤器，高Q干扰滤波器（HQ530/50）和一个565纳米（50％的传输）分色镜。荧光发射是分开使用分色镜和长通滤波器（HQ572LP）。中的INTEN入射光的密度调整使用中性密度过滤器。跨照明，明场观看的准备，提供了第二个100瓦的钨卤灯由一个0-55伏，1-10 A电源，不需要具备任何特殊属性的驱动。 </li><li>高速摄像头规格： 电子设计和NeuroCCD - SM相机的性能特点，已被描述详细 3 。简言之，它是一个冷却，分辨率低，精度高，速度的摄像头，使用背减薄，背照马可尼CCD39 - 01芯片（80x80像素）。数字化是14位，并以全帧速率为2 kHz。然而，对于特定的目的，可能会增加帧速率最高为10 kHz，通过各种分级组合或减少功能的像素数量。该相机具有极低的读出噪声（23电子在2 kHz; 9电子在1 kHz）的重要优势。此外，它的像素有一个比较大的井的深度（215000电子），允许在高帧频的光照强度适中。 </li><li>数据采集​​和分析软件： 光学实验控制Neuroplex，一个软件包从RedShirtImaging，作为内IDL平台（交互式数据语言）的应用程序，包括光学数据的采集，显示和分析所必需的多种功能。 </li><li>额外的相机： 作为仪器的组成部分，一个三目显微镜继电器到第二，高分辨率CCD相机连接到一个帧的采集编制的形象头分光镜。的形象，这是收购和储存，使用全球Lab图像/ 2软件，可以叠加到亚毫秒级的显示的时间分辨NeuroCCD - SM相机所拍摄的光信号产生的空间原点的精确地图光学记录电压信号。在我们的实验室，这台相机的图像采集卡驻留在不同的计算机上一个比一个负责数据采集功能。因此，图像文件从一台计算机传输到另一台，小的高清晰度图像的大小和位置的调整需要实现两个摄像头的完美登记前。 </li></ol>第7部分：光学实验<ol><li>每个功能的录音之前，我们收购的地区，从我们计划通过光学手段记录电活动（6.4）高清晰度黑白图像。这个形象，叠加到地图的高速摄像头的像素时，可以帮助我们确定的结构，是光信号源。 </li><li> Neuroplex（见6.3）在适当的系统配置，还可以控制快门，和触发电刺激。由于高速数据采集需要大量的计算机的电源，这是可取的，如果可能的话，使用一个大容量的内存和高容量的硬盘驱动器的专用计算机。然而，这是不平凡的。我们自己的系统，例如，已有数岁，员工是太大，无法在国家的最先进的的计算机数据采集板。作为一种妥协，我们通过和我们的光学系统在Dell Precision 390配备了500 GB的硬盘驱动器和4 GB的RAM。这台计算机，使我们能够保持我们年纪大了，但足够，A / D转换和D / A板，同时仍然提供足够的电力来运行我们的实验。 </li><li>一旦收购结束，Neuroplex可以显示在多个互补的格式结果，下面详细介绍其中一些： <ol class="lalpha"><li>在屏幕的一个区域，称为页面显示显示作为一个单独的帧的80X80高速相机拍摄的准备的原油形象。到这个第二个摄像头（在6.4和7.1的说明）收购的高清晰度图像的像素图的叠加，优化运营商的能力，以确定感兴趣的领域。 </li><li>当这个页面上显示的像素下鼠标控制选择时，Neuroplex会自动转换成注册为时间的函数由这些像素的光学变化幅度的痕迹，其光输出。 （例如，ΔF/ F随时间变化）。操作者可以决定这些痕迹是否应该反映单个像素的输出，或者，而编制的一个特定区域的多个位点的空间平均超过输出。 </li><li>这些数据也可以显示电影格式。 为了确保在个别实验运行中的所有信息是完全提取，但是，我们赞成光学数据的离线分析。代表的这一分析结果可用。 </li></ol></li></ol>第8部分：如何实现在光学实验中的技术​​质量钍E的光学实验技术，最终成功的衡量标准信号录音的信噪比。此外，一个良好的信号信噪比梗并不是一种顿悟，而是从一个旅程。在歌剧表演，最后的结果是由多重因素交互协同。因此，要实现一个技术上可靠的光学实验，它是必不可少的完善，独立，光学仪器，并为每个协议的各个步骤所需的技能。以下是一个潜在的麻烦地区名单，并为避免问题并不总是预计5的一些建议： <ol><li>噪声源 机械和光学稳定是至关重要的电压敏感染料记录电活动时，因为反映生理反应的微小变化（ΔF/ F）相对较小（&lt;10％/ 100毫伏在大多数肠道录音）和嘈杂的跟踪始终预示着一个坏的结果。因此，优化信号噪声比，它是必须消除，或者，至少，以尽量减少，表现最差的两个贡献：A）机械振动，和b）光源波动。 <ol class="lalpha"><li>机械振动产生的各种来源的多样化设置配置，建立稳定，机动设备，空气，噪声，流体的运动，或颗粒浮在洗澡的解决方案电流接近。一个良好的振动隔离系统，极端情况下的声窗帘（见，例如，在5.1中描述），有很大的不同，是非常值得他们的成本。此外，常识性的方法，如在实验中，或使用磁性底座小，可移动部件安装的解决方案，用于过滤，是相当有效的。 </li><li>光源是最稳定的准最大强度。因此，应该设置一个电源电流限制，并在必要时，通过插入中性密度滤光片在光路径，而不是通过降低电压降低光线强度。弧光灯本质上是嘈杂。钨卤灯泡或发光二极管更加稳定，应该是首选 6 。 </li></ol></li><li>动物的年龄 解剖应使用年轻动物的肠道，因为随着年龄的肠道toughens内的结缔组织，它变得更加难以分开两丛神经元的传入和传出的进程可能造成重大损失的个人组织层跨层边界和/或沿纵轴的肠道相当距离项目。最重要的咨询，研究神经节内连接时，可以忽略，当实验的目的是分析神经节间的电路或反射通路的激活。 </li><li>染料内化 重要的是要使用尽可能低的染料浓度，并尽量减少不必要的光线照射。 DI - 4 - ANEPPDHQ虽然已被证明比其他染料的光毒性要少得多，它就会内化，最终，作为该制剂的健康恶化。染料内化减少增加显着的背景荧光信号的信噪比。 </li><li>电极的维护 <ol class="jove_lalpha"><li>电极应审慎和彻底清洗后，每一个实验。在钨电极的技巧有机存款增加电极电阻，防止刺激。 </li><li>市售钨电极的绝缘层是非常微妙和容易损坏。在绝缘泄漏将分流，妥协的刺激效果，并危及实验的成功。 </li></ol></li><li>灌注危险 切勿将设置无人值守，同时灌注。如果在蠕动泵的流量和出流没有适当的平衡，两个灾难性事件可能发生： <ol class="lalpha"><li>洪水可能造成不可逆转的损害，在显微镜。 </li><li>在实验过程中可能会干出的编制。 </li></ol></li><li>软件的麻烦 它是必不可少的，以自己熟悉的软件来控制收购，以及分析。高速数据采集和/或创造非常大的文件往往驱动系统崩溃的边缘，Neuroplex，这是很无情的，无情的崩溃，除非其边界条件是尊重。在细微之处的记录，容易漏诊，当分析限于有限的地区的利益，往往显露出自己在电影显示的数据。 </li></ol>

<ol>
	<li>Costa, M. a. r. c. e. l. l. o., Furness, J. o. h. n. . B., Llewellyn-Smith, I. J., Johnson, L. e. o. n. a. r. d. . R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> in Physiology of the Gastrointestinal Tract, edited by Leonard R. , 1-1 (1987).</li><li>Obaid, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+patterns+of+activity+in+an+intact+mammalian+network+with+single-cell+resolution:+optical+studies+of+nicotinic+activity+in+an+enteric+plexus.">Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus.</a> J Neurosci. 19 (8), 3073-3073 (1999).</li><li>Obaid, A. L., Loew, L. M., Wuskell, J. P., Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+naphthylstyryl-pyridinium+potentiometric+dyes+offer+advantages+for+neural+network+analysis.">Novel naphthylstyryl-pyridinium potentiometric dyes offer advantages for neural network analysis.</a> J. Neurosci. Methods. 134 (2), 179-179 (2004).</li><li>Hirst, G. D., McKirdy, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+potentials+recorded+from+neurones+of+the+submucous+plexus+of+guinea-pig+small+intestine.">Synaptic potentials recorded from neurones of the submucous plexus of guinea-pig small intestine.</a> J Physiol (Lond). 249 (2), 369-369 (1975).</li><li>Salzberg, B. M., Barker, J., McKelvy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Methods+in+Cellular+Neurobiology.">Current Methods in Cellular Neurobiology.</a> , 139-139 (1983).</li><li>Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultra-stable+non-coherent+light+source+for+optical+measurements+in+neuroscience+and+cell+physiology.">An ultra-stable non-coherent light source for optical measurements in neuroscience and cell physiology.</a> Journal of Neuroscience Methods. 141, 165-16 (2005).</li><li>Obaid, A. L., Nelson, M. E., Lindstrom, J., Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+studies+of+nicotinic+acetylcholine+receptor+subtypes+in+the+guinea-pig+enteric+nervous+system.">Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system.</a> J Exp Biol. 208 (Pt 15), 2981-2981 (2005).</li><li>Obaid, A. L., Zou, D. J., Rohr, S., Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+with+single+cell+resolution+from+a+simple+mammalian+nervous+system:+Electrical+activity+in+ganglia+from+the+submucous+plexus+of+the+guinea-pig+ileum.">Optical recording with single cell resolution from a simple mammalian nervous system: Electrical activity in ganglia from the submucous plexus of the guinea-pig ileum.</a> Biol. Bull. 183, 344-344 (1992).</li><li>Salzberg, B. M., Davila, H. V., Cohen, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+impulses+in+individual+neurones+of+an+invertebrate+central+nervous+system.">Optical recording of impulses in individual neurones of an invertebrate central nervous system.</a> Nature. 246 (5434), 508-508 (1973).</li></ol>

该协议已被写入两个目标。第一个是说服其他调查，由于许多技术进步，在过去十年中，利用电压敏感染料的电气活动的光记录，已成为最强大的，可靠和可负担得起的完整的神经网络研究的方法之一，事实上，它可以很容易地实施，甚至在实验室有限的资源。第二个目标是作为一个独特的实验模型，以促进肠道的神经系统，在其中两个神经丛和生物输出的电气行为相关的分子和细胞活动。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Medium 199</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>M3769</td>
<td>With Earle&#x2019;s salts Without L-Glutamine, Phenol Red and Sodium Bicarbonate</td>
</tr>
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Dow Corning Corp., Midland, MI</td>
<td>184 silicone elastomer kit</td>
<td>&nbsp;</td>
<tr>
<td>Insect pins used for dissection</td>
<td>&nbsp;</td>
<td>F.S.T., Foster City, CA</td>
<td>26001-30</td>
<td>&nbsp;</td>
<tr>
<td>Dumont forceps</td>
<td>&nbsp;</td>
<td>F.S.T., Foster City, CA</td>
<td>11203-23 or 11200-33</td>
<td>&nbsp;</td>
<tr>
<td>Moria MC19/B Pascheff-Wolff Spring Scissors</td>
<td>&nbsp;</td>
<td>F.S.T., Foster City, CA</td>
<td>15371-92</td>
<td>&nbsp;</td>
<tr>
<td>Pins to hold the tissue during the experiment</td>
<td>&nbsp;</td>
<td>Seirin Corp., Shizuoka, Japan</td>
<td>Seirin Spinex</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase VII</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>C0773</td>
<td>&nbsp;</td>
<tr>
<td>Protease IX</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>P6141</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen, Carlsbad, CA</td>
<td>10439-016</td>
<td>&nbsp;</td>
<tr>
<td>PenStrep</td>
<td>&nbsp;</td>
<td>GIBCO, BRL</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine 100X</td>
<td>&nbsp;</td>
<td>GIBCO, BRL</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Nifedipine</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Di-4-ANEPPDHQ</td>
<td>&nbsp;</td>
<td>Molecular Probes, Eugene, OR</td>
<td>D36802</td>
<td>&nbsp;</td>
<tr>
<td>4-Channel Stimulus Generator</td>
<td>&nbsp;</td>
<td>Multi Channel System MCS GmbH, Reutlingen, Germany</td>
<td>2000 Series</td>
<td>&nbsp;</td>
<tr>
<td>Peristaltic pump</td>
<td>&nbsp;</td>
<td>Rainin, Woburn, MA</td>
<td>2-channel Dinamax RP-1</td>
<td>&nbsp;</td>
<tr>
<td>High-speed camera</td>
<td>&nbsp;</td>
<td>RedShirtImaging, LLC Decatur, GA</td>
<td>NeuroCCD-SM</td>
<td>&nbsp;</td>
<tr>
<td>1:10 demagnifier / relay lens</td>
<td>&nbsp;</td>
<td>Optem, Qioptiq Linos, Inc., Rochester, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Upright microscope</td>
<td>&nbsp;</td>
<td>Olympus Optical Co., Tokyo, Japan</td>
<td>BX61TRF</td>
<td>&nbsp;</td>
<tr>
<td>Gibraltar fixed stage with motorized X-Y translator</td>
<td>&nbsp;</td>
<td>Gibraltar, Burleigh Instruments, EXFO, Quebec, Canada</td>
<td>PSSG--BX2</td>
<td>&nbsp;</td>
<tr>
<td>Linear matrix electrodes</td>
<td>&nbsp;</td>
<td>FHC, Inc., Bowdoin, ME</td>
<td>Custom made</td>
<td>&nbsp;</td>
<tr>
<td>Active vibration-isolation table</td>
<td>&nbsp;</td>
<td>Halcyonics,Inc., Menlo Park, CA</td>
<td>Micro 60</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-low-ripple feedback stabilized power supply</td>
<td>&nbsp;</td>
<td>Kepco, Flushing, NY</td>
<td>ATE 75-15M</td>
<td>&nbsp;</td>
<tr>
<td>Heat filter</td>
<td>&nbsp;</td>
<td>Schott Optical Glass, Duryea, PA</td>
<td>KG-1</td>
<td>&nbsp;</td>
<tr>
<td>High-Q interference filter</td>
<td>&nbsp;</td>
<td>Chroma, Inc., Rockingham, VT</td>
<td>HQ530/50</td>
<td>&nbsp;</td>
<tr>
<td>Dichroic mirror</td>
<td>&nbsp;</td>
<td>Chroma, Inc., Rockingham, VT</td>
<td>565 nm (50% transmission)</td>
<td>&nbsp;</td>
<tr>
<td>Long-pass filter</td>
<td>&nbsp;</td>
<td>Chroma, Inc., Rockingham, VT</td>
<td>HQ572LP</td>
<td>&nbsp;</td>
<tr>
<td>Acoustic curtains</td>
<td>&nbsp;</td>
<td>Acoustical Surfaces, Inc.</td>
<td>BSC-25</td>
<td>&nbsp;</td>
<tr>
<td>Neuroplex</td>
<td>&nbsp;</td>
<td>RedShirtImaging, Inc., Decatur, GA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>IDL</td>
<td>&nbsp;</td>
<td>ITT Visual Information Solutions, Boulder, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>High-resolution CCD camera, with its own camera controller</td>
<td>&nbsp;</td>
<td>Hamamatsu Photonics, KK, Hamamatsu City, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Frame-grabber for high-resolution camera</td>
<td>&nbsp;</td>
<td>Data Translation, Marlboro, MA</td>
<td>DT3120K-1</td>
<td>&nbsp;</td>
<tr>
<td>Imaging software for high-resolution camera</td>
<td>&nbsp;</td>
<td>Data Translation, Marlboro, MA</td>
<td>Global Lab Image/2</td>
<td>&nbsp;</td>		</tbody>
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optical recording of electrical activity in guinea-pig enteric networks using voltage-sensitive dyes

肠神经系统（ENS），是一个自成体系的网络识别功能，能够执行复杂的行为，在隔离。丛局限于肠壁不同的飞机被安排在它的神经元（直径在10至25微米）<sup&gt; 1</sup&gt;肌间神经丛，可发现之间的纵向和圆形的肌肉层之间的环状肌肉层和黏膜黏膜下神经丛。由于这些丛效应系统（运输上皮细胞，内分泌细胞，免疫分子，血管平滑肌），也包含肠壁内，半完整的准备，可以解剖，保留不同的反射通路的各个组成部分。效应系统的行为是一致行动的黏膜下和肌间神经丛控制。因此，内部和之间的神经节，丛之间的沟通和突触相互作用的详细知识，了解正常的胃肠功能至关重要。 ENS的，作为一个完整的神经系统，是一个独特的实验模型，在其中一个可以相关的神经网络及其生理输出的电气特性的分子和细胞活动。由于其丛准二维组织，ENS的是特别适合于使用多个站点的光学记录技术，采用电压敏感染料的神经网络研究<sup&gt; 2,7,8,9</sup&gt;。我们将在这里说明一个相对较新naphthylstyryl吡啶染料（DI - 4 - ANEPPDHQ）使用<sup&gt; 3</sup提供了多种优势，它的前辈，包括光毒性非常低，速度缓慢的内化，以及出色的化学稳定性。在同一个摄像头，允许亚毫秒级的时间分辨率一起使用时，这种染料使我们能够监视视野中的所有神经元的电活动与100X倍率〜2.5微米的一个最大的空间分辨率。在较低的放大倍率（10X或20X），单细胞分辨率的牺牲是一个角度增益补偿，揭示间神经节电路的复杂性。

Watch this Scientific Journal Video about Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes at JoVE.com

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

该协议说明了电压敏感染料如何使豚鼠肠神经系统的神经丛，如完整的神经网络电活动的可调节分辨率的光学记录，范围从单细胞到多节后电路。

在豚鼠肠道网络，利用电压敏感染料光学记录的电活动

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

optical-recording-electrical-activity-guinea-pig-enteric-networks

Nanyang Technological University

Research

JoVE Journal

Biology

9.9K 次观看.  University of Pennsylvania-School of Medicine. 该协议说明了电压敏感染料如何使豚鼠肠神经系统的神经丛，如完整的神经网络电活动的可调节分辨率的光学记录，范围从单细胞到多节后电路。

视频: Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

科研

生物学

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

使用CyDye DIGE Fluor公司最小染料的细胞表面蛋白的选择性标签

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

质粒来源的DNA链置换盖茨实施化学反应网络

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

通过使用光稳定菁染料波长移的DNA杂交探针的合成

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

基于加权相关网络的不同栖息地根微生物群的差异

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...