这项工作是从美国国立卫生研究院的研究补助金（HL068915 HL088206）和在波士顿儿童医院的心脏传导基金捐款支持。

第1部分：准备的解决方案和离体心脏系统<ol><li>在上午的实验中，4.0 L是克雷布斯- Henseleit溶液准备如前所述[5，6] 。 </li><li> 11 mmol / L的2,3 - 丁二酮肟（BDM），溶解在1.0步骤1.1准备灌流转自克雷布斯Henseleit解决方案的大号。 </li><li>从在步骤1.1准备灌流克雷布斯- Henseleit一个额外的150毫升和5 mol / L的DI - 8 - ANEPPS（10毫摩尔/升，溶解在二甲基亚砜（DMSO）的股票稀释）混合。 </li><li>这3个方案被转移到水套式玻璃水库（Radnoti）是预先加热至41.0 ° C，并与淹没起泡使用0.2微米的过滤95％O 2（Radnoti）含氧; 5％的CO 2气体。 3股票水库的解决方案MASTERFLEX™L / S蠕动泵和低吸收硅胶管（科尔 - 帕默）输送壁挂式的Langendorff系统。 </li><li>大会之前的Langendorff系统玻璃器皿（Radnoti）认真洗净，用E - TOXA清洗试剂（Sigma - Aldrich公司）和0.1 M / L盐酸，100％的乙醇，蒸馏水彻底冲洗。 </li><li>非循环的Langendorff仪器被修建提供70毫米汞柱，从3个独立的含氧的水库，这是发自内心的分离与水套式加热线圈和泡沫陷阱的恒压（Radnoti）[ 7]。 3个独立的灌注线衔接上述两个3活塞的心，使我们能够精确地控制在1.1至1.3的步骤准备的解决方案交付。水套玻璃器皿MASTERFLEX PharMed BPT油管（科尔 - 帕默）系列连接，并加热到39 ° C的蒸馏水，采用两个环形（劳达）E100。通过铂固化硅胶管（科尔 - 帕默）连接压头水库的加热线圈和泡沫陷阱热损失在37 ° C灌流到达心脏。 </li></ol>第2部分：收获的大鼠心脏和设置Langendorff灌流<ol><li>深全身麻醉，诱导Lewis大鼠200-250克，100毫克/公斤氯胺酮及10毫克/公斤甲苯噻嗪注射到腹腔内。这种混合物中，我们添加500 U /公斤肝素，以防止在explantation过程中的凝血功能和心肌缺血。 </li><li>为了便于访问的心脏及大血管，前胸壁被删除。之后，周围组织仔细解剖，打开心包的麻袋。 </li><li>以下的下腔静脉的鉴定，此船是用5-0丝线（爱惜康）和整个心肺块explanted结扎。组织立即放置在冰冷的克雷布斯- Henseleit一个50 mL烧杯中，在冰中的解决方案。 </li><li>迅速查明升主动脉和周围组织的解剖。一个大小合适的套管（哈佛器械）被插入到主动脉，照顾，以避免中断通过插入到主动脉根部太远插管灌注冠状动脉的义务。 5-0丝线（爱惜康）升主动脉插管是安全。 </li><li>大鼠心脏的Langendorff仪器，然后放置在没有引入气泡套管。逆行冠状血管灌注的温暖，含氧克雷布斯Henseleit步骤1.6中所描述的压力头解决方案现已确定。 </li><li>现在删除额外的组织，包括肺，心灌流20分钟，以允许功能的恢复和稳定的节奏。在这段时间内，一个非常薄的热电偶温度探头（科尔 - 帕默）引入左心室腔，并在5-0普理灵缝合（爱惜康）缝合。在探头连接到热控制器（从DIGI - SENSE），以确保心脏的温度保持在37 °调整水循环泵的设置，从心尖灌流淋漓的运动是最大限度地降低污水中的插座放置一块纱布。 </li></ol>第3部分：心脏负荷电位染料和收购电图及光信号<ol><li> DI - 8 - ANEPPS（Invitrogen）的加载切换到含有荧光染料使用活塞克雷布斯Henseleit混合灌注线入心。此外，18G套管的左侧和/或右心房和额外的50毫升的染料溶液放入，慢慢进入每个商会管理，因为他们没有足够的灌注通过冠状动脉介绍染料。 </li><li>在装载过程中，3心电图导联（哈佛器械），轻轻地放在心，面对的不是用于映射的光学表面。电极定位后左心室心尖部分，N O 2的左心房上，和N N O 1 <su<html> P&gt; O 3对主动脉根部（图1）作为参比电极。随后心房和心室电图信号放大，数字化，并使用该软件（RedShirt影像）（图2）的光信号一起显示。示波器（泰克模式的TDS 1002）也可用于可视化实时体表心电图小号，以保证足够的起搏。 </li><li>心电图放大器（雨果萨克斯Elektronik公司）设置：高通滤波器：0.1赫兹 低通滤波器：150赫兹</li><li> CMOS摄像头（RedShirt成像）和macroscope使用XYZ的调整定位，使心脏表面的重点和中心在收购框架。摄像机和光学系统安装在振动隔离表（减号k技术），以尽量减少共振频率。同时，同轴的起搏电极（哈佛器械）控制的一个孤立的，S48电刺激装置（草），是摆在右心房和心脏的节奏在每分钟300次（图1）。 </li><li>电刺激（草）的设置： 速率：每秒5脉冲 延迟：0.2毫秒 时间：2毫秒 电压：6-12 V 模式：重复 脉冲：单</li><li>对于缺乏收缩运动伪影的光学录音，心脏需要机电耦合。为此，我们再次切换灌注线克雷布斯Henseleit解决方案，其中包含11毫摩尔/大号的BDM。采集之间，心是十足的克雷布斯- Henseleit灌注，以帮助维护编制的可行性。 </li><li>记录参数设置，使用下面的收购设置软件（RedShirt影像）： 配置：2000赫兹; 128x128像素阵列 帧间隔：0.5毫秒 相机放大器增益：5倍 片上增益：8X - 12Me井 快门速度：500毫秒延迟 张数：4000 时间：2000毫秒</li><li>所有会议室和设备的灯都关闭或屏蔽，以消除在录制过程中的背景噪音。 LED灯亮起，仅在光学记录的心，以减少光漂白和染料的毒性。通过控制面板提供一个D -在电脑板（RedShirt成像）的方式与一个5 V脉冲光源的快门是控制。 </li></ol>第4部分：使用RedShirt图像处理软件，分析采集信息<ol><li>采集，数据处理使用不同的过滤器设置。除了调整时，乐队停止/通滤波器，这是遗留的边界在44.0和98.0正确的边界设置，我们一般使用默认设置。随后记录的信息处理和生成（RedShirt影像）的影片。 </li><li>从一个采集数据对应的地方在心脏表面上的16384站点超过2秒内的电激活。该软件允许直接与彼此之间以及与心房和心室电图的录音相比，这些地方的信号。数据，然后通过当地的电气激活映射的颜色和渲染动画显示的时空心脏表面上的电激活信息可视化。要创造这样一个动画，我们使用的软件： <ul><li>时间上和/或空间的数据进行筛选</li><li>选择一个动画的开始和结束时间</li><li>地图颜色的基础上休息的每个像素的光线强度的光信号</li><li>心的图片叠加产生的颜色数据</li><li>生成动画</li></ul></li></ol>第5部分：代表结果如果灌注心脏准备在录制过程中一动不动，光信号显示DI - 8 - ANEPPS排放强度的变化所涉及的每一个像素点不同的高峰之一。 （图3和4）相应的电影表现出一个激励整个的心，以及同时收购（图2）电图录音外膜表面传播的波前。 <img src="/files/ftp_upload/1138/Figure3.jpg" alt="图1" /> 图1。一个的Langendorff灌注心脏准备的照片描绘右心房起搏电极的位置和心电图导致在步骤3.2中所述。 <img src="/files/ftp_upload/1138/Figure4thumb.jpg" alt="图2" border="0" /> 图2代表的光信号从一个刘易斯灌流大鼠心脏电图的录音。 A显示了一个用于光学成像的心外膜表面的形象。选择显示在荧光发射在B组随时间的​​变化像素的位置，是由彩色箭头表示。电图信号显示在C组，表明心房激活和心室信号对应的淡蓝色线与红色线。请ve.com/files/ftp_upload/1138/Figure4.jpg“&gt;点击这里看的这个数字大的版本。

<ol>
	<li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ Res. 95 (1), 21-33 (2004).</li><li>Hucker, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Images+in+cardiovascular+medicine.+Optical+mapping+of+the+human+atrioventricular+junction.">Images in cardiovascular medicine. Optical mapping of the human atrioventricular junction.</a> Circulation. 117 (11), 1474-147 (2008).</li><li>Entcheva, E., Bien, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+optical+mapping+of+excitation+in+cardiac+cell+networks+with+ultra-high+spatiotemporal+resolution.">Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution.</a> Prog Biophys Mol Biol. 92 (2), 232-257 (2006).</li><li>Entcheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+electrical+activity+in+cardiac+cells+using+an+all-solid-state+system.">Fluorescence imaging of electrical activity in cardiac cells using an all-solid-state system.</a> IEEE Trans Biomed Eng. 51 (2), 333-341 (2004).</li><li>Stamm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+endotoxin-induced+alterations+in+myocardial+calcium+handling:+obligatory+role+of+cardiac+TNF-alpha.">Rapid endotoxin-induced alterations in myocardial calcium handling: obligatory role of cardiac TNF-alpha.</a> Anesthesiology. 95 (6), 1396-1405 (2001).</li><li>Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+conduction+through+engineered+tissue.">Cardiac conduction through engineered tissue.</a> Am J Pathol. 169 (1), 72-85 (2006).</li><li>Skrzypiec-Spring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium.">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> J Pharmacol Toxicol Methods. 55 (2), 113-126 (2007).</li><li>Fedorov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+blebbistatin+as+an+excitation-contraction+uncoupler+for+electrophysiologic+study+of+rat+and+rabbit+hearts.">Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts.</a> Heart Rhythm. 4 (5), 619-626 (2007).</li></ol>

机构动物护理和使用委员会在波士顿儿童医院的规定的准则和法规的规定进行了动物实验。

麻醉大鼠的心去除，必须快速进行，以避免心肌缺血。如果发生缺血或冠状动脉灌注不足，心脏可能会发展性心律失常，并可能成为心肌梗死。此外，这些心会显示信息录音和后续分析的荧光发射不足。加载染料之前，需要充分克雷布斯- Henseleit溶液灌注心肌细胞，建立和保持一个稳定的电脉冲的生理电解质的环境。准确的灌流准备也是必要的，以保持适当的的器官的活力和功能。电解质浓度或?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CardioCMOS-SM128f</td>
<td>Equipment</td>
<td>RedShirt Imaging, Decatur, GA 30030 USA </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CardioPlex Software</td>
<td>Equipment</td>
<td>RedShirt Imaging, Decatur, GA 30030 USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LUXEON LED Light Source 460-490 nm </td>
<td>Equipment</td>
<td>Lumileds Lighting, US, LLC, San Jose, CA 95131 USA</td>
<td>LXHL-PB02</td>
<td>&nbsp;</td>
<tr>
<td>ECG Amplifier Type 689 Hugo Sachs Elektronik</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>730149</td>
<td>&nbsp;</td>
<tr>
<td>Dichroic Mirror 505 nm</td>
<td>Equipment</td>
<td>Semrock, Rochester, NY 14624 USA</td>
<td>FF505-SDi01-25x36</td>
<td>&nbsp;</td>
<tr>
<td>Emission Filter 605 nm Long Pass</td>
<td>Equipment</td>
<td>SciMedia, Costa Mesa, CA 92626 USA </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>THT Sideways</td>
<td>Equipment</td>
<td>SciMedia, Costa Mesa, CA 92626 USA</td>
<td>25 BM-8</td>
<td>&nbsp;</td>
<tr>
<td>Mini Ball Joint Holder</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>BS4 73-0177</td>
<td>&nbsp;</td>
<tr>
<td>Small Stimulation Electrode Set</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>BS4 73-0160</td>
<td>&nbsp;</td>
<tr>
<td>BM-6 Benchtop Vibration Isolation Platform</td>
<td>Equipment</td>
<td>Technology Inc., Inglewood, CA 90301</td>
<td>25 BM-6</td>
<td>&nbsp;</td>
<tr>
<td>Monopolar ECG Electrode</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>BS4 73-0200</td>
<td>&nbsp;</td>
<tr>
<td>Roller Pump SCI 400</td>
<td>Equipment</td>
<td>Watson-Marlow Bredel Inc., Wilmington, MA 01887 USA</td>
<td>401U/D1</td>
<td>&nbsp;</td>
<tr>
<td>Roller Pump MasterFlex Easy Load II</td>
<td>Equipment</td>
<td>Cole Parmer, Vernon Hills, Illinois 60061 USA</td>
<td>Model 77201-60</td>
<td>&nbsp;</td>
<tr>
<td>Tubing Marprene #14</td>
<td>Equipment</td>
<td>Watson-Marlow Bredel Inc., Wilmington, MA 01887 USA</td>
<td>902.0016.016</td>
<td>&nbsp;</td>
<tr>
<td>MasterFlex Tubing</td>
<td>Equipment</td>
<td>PharMed, Westlake, OH 44145 USA</td>
<td>06485-25</td>
<td>&nbsp;</td>
<tr>
<td>S48 Square Pulse Stimulator</td>
<td>Equipment</td>
<td>Grass Technologies, West Warwick, RI 02893 USA</td>
<td>Model S48</td>
<td>&nbsp;</td>
<tr>
<td>SIU5 RF TRANSFORMER ISOLATION UNIT</td>
<td>Equipment</td>
<td>Grass Technologies, West Warwick, RI 02893 USA</td>
<td>Model SIU5</td>
<td>&nbsp;</td>
<tr>
<td>5 Liter Water Jacketed Reservoir</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>120142-5</td>
<td>&nbsp;</td>
<tr>
<td>2 Liter Water Jacketed Reservoir</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>120142-2</td>
<td>&nbsp;</td>
<tr>
<td>0.5 Liter Water Jacketed Reservoir</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>120142-0</td>
<td>&nbsp;</td>
<tr>
<td>0.25 Liter Water Jacketed Reservoir</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA </td>
<td>120142-025</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Heating Coil</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>158822</td>
<td>&nbsp;</td>
<tr>
<td>Compliance Bubble Trap</td>
<td>Equipment</td>
<td>Radnoti Glass Technology Inc., Monrovia CA 91016 USA</td>
<td>130149</td>
<td>&nbsp;</td>
<tr>
<td>Luer Disconnect Cannula</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>72-1444</td>
<td>&nbsp;</td>
<tr>
<td>3-Way stopcock, FLL to MLT, No Port Covers</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>BS4 72-2630</td>
<td>&nbsp;</td>
<tr>
<td>Thermocouple Thermometer</td>
<td>Equipment</td>
<td>Cole Parmer, Vernon Hills, Illinois 60061 USA</td>
<td>WU-91100-40</td>
<td>&nbsp;</td>
<tr>
<td>Ultra Fine IT-Series Flexible Microprobe </td>
<td>Equipment</td>
<td>PhysiTemp Instruments Inc., Clifton, NJ 07013 USA</td>
<td>IT-24P</td>
<td>&nbsp;</td>
<tr>
<td>Oscilloscope Tektronix TDS 1002</td>
<td>Equipment</td>
<td>Tektronix Inc., Beaverton, OR 97005 USA</td>
<td>TDS 1002B</td>
<td>&nbsp;</td>
<tr>
<td>2,3-Butanedione monoxime </td>
<td>Reagent</td>
<td>Sigma, St. Louis, MO 63132 USA</td>
<td>B0753</td>
<td>&nbsp;</td>
<tr>
<td>Ketamine HCl</td>
<td>Reagent</td>
<td>Hospira Inc., Lake Forest, IL 60045 USA</td>
<td>RL-0065</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>Reagent</td>
<td>Lloyd Inc., Iowa 51601 USA</td>
<td>LB15705A</td>
<td>&nbsp;</td>
<tr>
<td>E-TOXA-CLEAN&reg;</td>
<td>Reagent</td>
<td>Sigma, St. Louis, MO 63132 USA </td>
<td>E9029</td>
<td>&nbsp;</td>
<tr>
<td>Di-8-ANEPPS</td>
<td>Reagent</td>
<td>Invitrogen, Carlsbad, CA 92008 USA</td>
<td>D-3167</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

optical mapping of langendorff-perfused rat hearts

电压敏感的荧光染料的心脏表面的光的映射已成为一个重要的工具，以调查在实验模型电励磁，在量程范围从细胞培养到整个器官<sup&gt; [1，2]</sup&gt;。几乎立即可使用状态的最先进的的光学成像系统，在正常心律或心律失常的启动和维护整个动作电位的产生和传播的可视化<sup“[1]</sup&gt;。最新的市售系统可以提供过高的时空分辨率的信息和最初开发定制的设备为基础，以克服由更传统的电方法施加的障碍<sup“[1]</sup&gt;。在高分辨率和高速互补金属氧化物半导体（CMOS）摄像头和强烈的光亮，发光二极管（LED）以及电压敏感染料，光学，和过滤器的进步已经开始使电气信号采集心血管疾病的细胞生物学家谁更习惯于用显微镜工作的实际。虽然新一代的CMOS摄像头，可以获取10000帧每秒16,384像素阵列上，根据类型的样品制备，长期形成的荧光采集技术，如光电二极管阵列，激光扫描系统，冷却电荷耦合器件（CCD ）相机仍然有一些明显的优势方面的动态范围，信号的信噪比和量子效率<sup&gt; [1，3]</sup&gt;。在本研究中，Lewis大鼠心脏灌流<em&gt;体外</em&gt;与一个晶体灌流（克雷布斯Henseleit溶液）在37 °上修改的Langendorff器具彗星。经过20分钟的稳定期，心中间歇灌注11 mmol / L的2,3 - 丁二酮肟，以消除收缩在图像采集相关的议案。对于光的映射，我们满载着心中的快速反应电位探头DI - 8 - ANEPPS<sup“[4]</sup&gt;（5μmol/ L的），并简要475 ± 15 nm的激发光照亮的准备。在一个典型的2第二期照明，高速CMOS摄像头连接到一个水平macroscope&gt;从心脏编制发出的605纳米的光成像。对于本演示，心中节奏在每分钟300次同轴电极连接到一个孤立的电刺激单元。同时双极电图的录音被收购，分析与使用现成可用的软件的电压信号。在这种方式下，可以可视化的Langendorff灌流大鼠心脏表面上的动作电位和电图信号注册。

Watch this Scientific Journal Video about Optical Mapping of Langendorff-perfused Rat Hearts at JoVE.com

Optical Mapping of Langendorff-perfused Rat Hearts

本文介绍了高时空分辨率技术的光学图像行动的Langendorff灌注大鼠的心，用一个电位染料（DI - 8 - ANEPPS）表面上的潜在流动。

光学测绘的Langendorff灌注大鼠心脏

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

optical-mapping-of-langendorff-perfused-rat-hearts

Research

JoVE Journal

Biology

20.7K 次观看.  Children's Hospital Boston and Harvard Medical School. 本文介绍了高时空分辨率技术的光学图像行动的Langendorff灌注大鼠的心，用一个电位染料（DI - 8 - ANEPPS）表面上的潜在流动。

视频: Optical Mapping of Langendorff-perfused Rat Hearts

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 ...

科研

生物学

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

光镜下的主要组成部分

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

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 ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

单细胞在斑马鱼的命运映射

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

细胞标记和注射胚胎发展小鼠心脏

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

培养HL-1心房肌细胞单层的电压和钙双通道光映射

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

的内肌浆网钙光学标测<sup&gt; 2+</sup&gt;和的Langendorff灌注兔心脏跨膜电位

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.