国立卫生研究院拨款R01 HL085369，HL067322，HL082729，EB008999

1。制备<ol><li>准备新鲜的Tyrodes“解决方案（毫米，128.2氯化钠，氯化钙1.3，4.7氯化钾，1.19的NaH 2 PO 4，1.05，20.0 MgCl 2的碳酸氢钠 3和11.1葡萄糖） 。为了加快解决方案的日常准备，提前准备两只股票的解决方案和存储在4 ° C冰箱：（1）股票我（g/2L，374.6氯化钠，9.56，8.21 17.52氯化钙，氯化钾的NaH 2 PO 4氯化镁2）和（2）股份II（在g/2L碳酸氢钠3），84.01，10.67。为了使Tyrodes的解决方案一个实验以足够的2L我股票，股票二80毫升，和4G的葡萄糖80毫升去离子水和混合1840mL。 </li><li>准备染料和uncouplers股票的解决方案：（1）兴奋收缩解偶联剂blebbistatin原液（Tocris生物科学2mg/mL的解决方案，在DMSO）;（2）电压敏感染料DI - 4 - ANEPPS原液（Invitrogen公司，为1mg/ml （3）解决方案，在DMSO）;电压敏感染料相对湿度237原液（Invitrogen公司1.25mg/ml在DMSO溶液）;（4）钙指标Rhod - 2AM原液（Invitrogen公司，1mg/ml的DMSO溶液）。一个单一的兔实验大约需要30μLDI - 4 - ANEPPS原液，30μLRH237原液，200μL的Rhod - 2AM原液。为了避免反复冻融，我们储存在-20 ° C 100微升等分DI - 4 - ANEPPS其他染料也存放在-20 ° C。储存在4 °彗星冰箱溶解blebbistatin。 </li><li>收获之前到2L的瓶子的心脏，转让Tyrodes的解决方案，并放入水浴（尔科技）的溶液温度保持在37 ° C。解决的办法是含氧95％O 2 - 5％的CO 2。 pH值保持在7.35 ± 0.05调整血氧水平。解决的办法是发给在Langendorff灌流系统，是由尼龙网过滤器（孔径：11μm，微孔）过滤，置于前插管灌注线。 </li><li>准备收获之前的心脏监测设备。一个压力传感器（WPI）是用来监视在实验过程中的主动脉压力。压力传感器的基准调整时心脏灌注系统没有连接到零毫米汞柱。伪心电图电极放置在近似铅I，II和III Einthoven三角心电图室。 </li></ol> 2。收获和灌注兔心<ol><li>修复兔在兔限位。安乐死兔静脉注射戊巴比妥钠（50毫克/公斤），与2000 U肝素。当兔子是完全安乐死，这是缺乏痛觉反射，迅速打开胸腔，心脏和肺部切除。 </li><li>请在升主动脉主动脉弓分支前的上端切。从升主动脉冲洗出来的空气，然后迅速cannulate 16号套管，先前已连接到一个泡沫捕手，这是非常重要的，保持空气冠状动脉的心脏。一旦心脏是逆行灌注在一个非循环的Langendorff灌流系统，削减迅速打开心包。 </li><li>取出肺，气管，脂肪和结缔组织，而血灌注冲洗。 </li><li>很重要的！一个硅胶管（〜3cm长，直径和2mm）通过肺静脉和二尖瓣进入左心室（LV）的插入，并保持在整个实验。这管释放被困在LV的解决方案。没有流通小时Lan​​gedorff在机械固定的心脏灌注实验期间，它可能会导致在LV腔严重缺血，产生心律失常。 </li><li>移动光学测绘仪器与套管循环的Langendorff灌注系统的心脏。 </li></ol> 3。进行实验使用的全景光学测绘系统<ol><li>放置在一个特制的六边形室的心脏，连接导管灌注系统。调整灌注泵的流量保持动脉压在60 ± 5毫米汞柱。监视器导致我伪心电图。保持pH值约为7.35 ± 0.05。 </li><li>关掉室内的灯光，因为紫外线和低端（450-490纳米）的频谱的可见部分的 17 photoinactivated blebbistatin 。通过注射套管上方的气泡捕手口慢慢注入blebbistatin原液。慢慢注入0.1毫升blebbistatin每推注。前等待下一次注射压力稳定。 </li><li>轻轻地放置到特定的位置，以你的实验设计的起搏电极。 </li><li>重点从三个磨砂玻璃位于平面图像的每个光电二极管阵列（PDA）的心脏图像均匀分布心脏周围的角度。调整位置的套管和每个PDA和心脏之间的距离，以适应心脏在所有三个掌上电脑领域的视图。以每个集中在磨砂玻璃形象的图片。 </li><li>慢慢注入10〜20μLDI - 4 - ANEPPS通过原液注射到灌注液中的气泡陷阱端口。采取光学录音之前，请等待1〜3分钟。 </li><li>第一张唱片，打开绿色的LED灯（没有激发滤光片，LED泛光灯，Lumileds公司），同时采取与定制的数据采集系统连接三个掌上电脑 ，5光学录音和关闭LED灯。检查从不同的三个掌上电脑的所有像素的信号质量。如果注意到在光学动作电位的运动伪影，添加0.1〜悬液0.2ml blebbistatin原液。如果信号信噪比低，另外5μLDI - 4 - ANEPPS原液添加。 </li><li>完成的功能研究设计的实验方案。再额外5μLDI - 4 - ANEPPS原液染色心脏，如果实验过程中，由于漂白或冲刷恶化的信号。 </li><li>房间的灯打开后，完成的功能的研究。从36个均匀分布的角度，对心脏的图片。这是通过旋转10 °的一个步骤，与固定在一个PDA的位置数码相机的心脏。 </li><li>室取出的心脏。漏所有的解决方案。 DI水，70％的试剂酒精，并再次DI水的顺序清洗灌注系统。 </li><li>数据分析包括从36个数码照片，登记到重建的几何表面的光信号，和量化的动作电位时程（APD），传导速度（CV），相位等 6个心脏几何重建</li></ol> 4。使用双绘图系统进行实验<ol><li> （第2部分后继续）放置在一个玻璃容器（Radnoti）空心的心脏和连接导管灌注系统。牵制的硅，在心室心尖部和心房腔底部的心脏。 </li><li>关掉室内的灯光。慢慢注入的blebbistatin股票解决方案（15〜20分钟达到10μM）前通过注射口灌注导管固定的心脏。 </li><li>把一个塑料培养皿中，心外膜表面或以上其他玻璃窗口覆盖，降低溶液的表面的议案。 </li><li>聚焦在两个CMOS摄像头的双绘图系统在相同的视野（终极- L SciMedia）。发射荧光是由一个分色镜（635nm的截止，欧米茄光）分离，由一个700nm的长通滤波器（Thorlabs）为电压信号，由三十零分之五百九十零纳米带通滤波器（欧米茄光学）钙信号的过滤。 </li><li> AIM两个卤素灯（新港奥丽尔仪器，斯特拉特福德，CT; SciMedia，科斯塔梅萨，加利福尼亚）的光导对测绘领域的观点，以达到均匀的照明。用于激励过滤器（四十〇分之五百三十一纳米，SemRock）。 </li><li>染色电压敏感染料相对湿度237原液通过注射口（10〜30μL）的心脏。 </li><li>组合聚醚F - 127的Rhod上午02时（悬液0.2ml）原液（Invitrogen公司，1:1的混合物）。 1分钟超声在水浴sonicator。通过泡沫捕手的端口注入混合物。等待约20分钟，让Rhod - 2 -酯化上午开始之前映射。 </li><li>对于一个记录，关闭灌流泵，以避免在表面的解决方案的议案;打开的激发光源（卤素灯）;采取使用这两款相机连接到数据采集系统（终极- L SciMedia）的光学录音关闭激发光;打开灌流泵。检查光信号的质量。重新染色的组织，如果必要的。 </li><li>完成的一项研究设计的实验方案的其余部分。 </li><li>打开室内的灯光，并采取包含视野的心脏的照片。室取出的心脏。漏所有的解决方案。在序列的试剂70％酒精（尔科技），去离子水，DI水冲洗灌注系统。 </li><li>数据分析包含的APD，简历，钙瞬变时间（CaTD），AP向右上挑和猫上升，钙瞬变的上升时间，和一个单指数拟合的CAT衰减的时间常数之间的延迟测量。 </li></ol>代表性的成果： <img src="/files/ftp_upload/3160/3160fig1.jpg" alt="图1"/> 图1。一个的Langendorff灌注兔实验使用的全景光学测绘系统的代表结果。 （一）在兔心脏和重建，形成一个立体的网状网格表面的兔子心脏几何的前视图。 （B）T他解开心外膜表面颜色编码的快速心律失常发作时显示为红色的眼波相（相平面分析18获得）。 （三）光学行动从五个地点周围标记面板B.（四）八激活眼波（红色）传播的快照在一个稳定的折返性心律失常的周期由1-5的阶段奇异的潜力录音。波前圆顺时针围绕一个阶段的奇点，这是在心脏的前表面可见。复极的颜色（蓝色）是部分透明，让后波前是可见的（例如，在80毫秒，100毫秒，而120毫秒）。此折返性心律失常的电影中提供的补充视频1。几何重建，信号登记，计算相图，和表面解缠的方法是在细节别处6描述。 <img src="/files/ftp_upload/3160/3160fig2.jpg" alt="图2"/> 图2：从一个的Langendorff灌注兔心脏的实验使用的双绘图系统（动作电位和钙瞬变的同时映射）代表结果。 （一）心脏的前表面的黑点所涵盖的映射字段。 （B）从一个站点的录音特写。 （三）样品的痕迹（蓝色）的动作电位和钙瞬变阵列标记面板A.注意，并非所有的像素录音显示和空间分辨率为200μm的黑点均匀分布地点（红色）。

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	<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, 21-33 (2004).</li><li>Panfilov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+heart+size+a+factor+in+ventricular+fibrillation?+Or+how+close+are+rabbit+and+human+hearts?.">Is heart size a factor in ventricular fibrillation? Or how close are rabbit and human hearts?.</a> Heart Rhythm. 3, 862-864 (2006).</li><li>Maier, L. S., Bers, D. M., Pieske, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+Ca(2+)-handling+and+sarcoplasmic+reticulum+Ca(2+)-content+in+isolated+rat+and+rabbit+myocardium.">Differences in Ca(2+)-handling and sarcoplasmic reticulum Ca(2+)-content in isolated rat and rabbit myocardium.</a> J Mol Cell Cardiol. 32, 2249-2258 (2000).</li><li>Bray, M. A., Lin, S. F., Wikswo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+surface+reconstruction+and+fluorescent+visualization+of+cardiac+activation.">Three-dimensional surface reconstruction and fluorescent visualization of cardiac activation.</a> IEEE Trans Biomed Eng. 47, 1382-1391 (2000).</li><li>Qu, F., Ripplinger, C. M., Nikolski, V. P., Grimm, C., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+panoramic+imaging+of+cardiac+arrhythmias+in+rabbit+heart.">Three-dimensional panoramic imaging of cardiac arrhythmias in rabbit heart.</a> J Biomed Opt. 12, 044019-044019 (2007).</li><li>Lou, Q., Ripplinger, C. M., Bayly, P. V., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+panoramic+imaging+of+epicardial+electrical+activity.">Quantitative panoramic imaging of epicardial electrical activity.</a> Ann Biomed Eng. 36, 1649-1658 (2008).</li><li>Kay, M. W., Amison, P. M., Rogers, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+surface+reconstruction+and+panoramic+optical+mapping+of+large+hearts.">Three-dimensional surface reconstruction and panoramic optical mapping of large hearts.</a> IEEE Trans Biomed Eng. 51, 1219-1229 (2004).</li><li>Li, W., Ripplinger, C. M., Lou, Q., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+monophasic+shocks+improve+electrotherapy+of+ventricular+tachycardia+in+a+rabbit+model+of+chronic+infarction.">Multiple monophasic shocks improve electrotherapy of ventricular tachycardia in a rabbit model of chronic infarction.</a> Heart Rhythm. 6, 1020-1027 (2009).</li><li>Ripplinger, C. M., Lou, Q., Li, W., Hadley, J., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Panoramic+imaging+reveals+basic+mechanisms+of+induction+and+termination+of+ventricular+tachycardia+in+rabbit+heart+with+chronic+infarction:+implications+for+low-voltage+cardioversion.">Panoramic imaging reveals basic mechanisms of induction and termination of ventricular tachycardia in rabbit heart with chronic infarction: implications for low-voltage cardioversion.</a> Heart Rhythm. 6, 87-97 (2009).</li><li>Efimov, I. R., Rendt, J. M., 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+maps+of+intracellular+[Ca2+]i+transients+and+action-potentials+from+the+surface+of+perfused+guinea-pig+hearts+(abstract).">Optical maps of intracellular [Ca2+]i transients and action-potentials from the surface of perfused guinea-pig hearts (abstract).</a> Circulation. 90, 632-632 (1994).</li><li>Choi, B. R., 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=Simultaneous+maps+of+optical+action+potentials+and+calcium+transients+in+guinea-pig+hearts:+mechanisms+underlying+concordant+alternans.">Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.</a> J Physiol. 529, 171-188 (2000).</li><li>Fast, V. G., Ideker, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+optical+mapping+of+transmembrane+potential+and+intracellular+calcium+in+myocyte+cultures.">Simultaneous optical mapping of transmembrane potential and intracellular calcium in myocyte cultures.</a> J Cardiovasc Electrophysiol. 11, 547-556 (2000).</li><li>Laurita, K. R., Singal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+action+potentials+and+calcium+transients+simultaneously+from+the+intact+heart.">Mapping action potentials and calcium transients simultaneously from the intact heart.</a> Am J Physiol Heart Circ Physiol. 280, 2053-2060 (2001).</li><li>Choi, B. R., Burton, F., 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=Cytosolic+Ca2++triggers+early+afterdepolarizations+and+Torsade+de+Pointes+in+rabbit+hearts+with+type+2+long+QT+syndrome.">Cytosolic Ca2+ triggers early afterdepolarizations and Torsade de Pointes in rabbit hearts with type 2 long QT syndrome.</a> J Physiol. 543, 615-631 (2002).</li><li>Hwang, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+and+vulnerability+to+fibrillation+and+defibrillation+in+Langendorff-perfused+rabbit+ventricles.">Intracellular calcium and vulnerability to fibrillation and defibrillation in Langendorff-perfused rabbit ventricles.</a> Circulation. 114, 2595-2603 (2006).</li><li>Lou, Q., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+susceptibility+to+alternans+in+a+rabbit+model+of+chronic+myocardial+infarction.">Enhanced susceptibility to alternans in a rabbit model of chronic myocardial infarction.</a> Conf Proc IEEE Eng Med Biol Soc. , 4527-4530 (2009).</li><li>Kolega, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxicity+and+photoinactivation+of+blebbistatin+in+UV+and+visible+light.">Phototoxicity and photoinactivation of blebbistatin in UV and visible light.</a> Biochem Biophys Res Commun. 320, 1020-1025 (2004).</li><li>Bray, M. A., Wikswo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+in+phase+plane+analysis+for+nonstationary+reentrant+cardiac+behavior.">Considerations in phase plane analysis for nonstationary reentrant cardiac behavior.</a> Phys Rev E Stat Nonlin Soft Matter Phys. 65, 051902-05 (2002).</li><li>Fast, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+action+potentials+using+voltage-sensitive+dyes.">Recording action potentials using voltage-sensitive dyes.</a> Practical methods in cardiovascular research. , 233-255 (2005).</li><li>Knisley, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+voltage+changes+during+unipolar+stimulation+of+rabbit+ventricle.">Transmembrane voltage changes during unipolar stimulation of rabbit ventricle.</a> Circ Res. 77, 1229-1239 (1995).</li><li>Bishop, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+photon+scattering+in+optical+signal+distortion+during+arrhythmia+and+defibrillation.">The role of photon scattering in optical signal distortion during arrhythmia and defibrillation.</a> Biophys J. 93, 3714-3726 (2007).</li><li>Efimov, I. R., Fedorov, V. V., Joung, B., Lin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cardiac+pacemaker+circuits:+methodological+puzzles+of+the+sinoatrial+node+optical+mapping.">Mapping cardiac pacemaker circuits: methodological puzzles of the sinoatrial node optical mapping.</a> Circ Res. 106, 255-271 (2010).</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, 619-626 (2007).</li></ol>

没有利益冲突的声明。

根据我们的经验，一个成功的Langendorff灌注兔心脏实验的关键，包括精心准备Tyrodes的解决方案，快速收获的心脏，维护良好的灌注压，和适当的pH值在灌注系统的含氧解决方案。为了记录尽可能高的信号信噪比的信号，我们需要考虑的因素包括光源，光学过滤器，聚焦光学系统，光电探测器等19。这些方面的详细讨论其他地方 19 。幼兔（4-5个月;重量：年龄7-9磅），可以用来避免心外膜脂肪，从而减少了信号，光信号的信噪比。 记录每个像素的信号是从组织体积所发出的光的加权融合。本组织体积的深度取决于所用染料的激发和发射波长。对于DI - 4 - ANEPPS，作为一个例子，估计穿透深度是在兔子心脏20300μm。因此，光信号的解释应谨慎时，当地的电气功能的异质性窦房结，房室结，并在室性心律失常1,21,22。 与电极记录相比，光的映射技术的一个限制，往往是由心脏收缩造成的运动伪影distored的光学动作电位复极相。可以用来减少工件机械约束，但不能完全消除它。相比之下，药理激发收缩uncouplers有效地​​消除运动伪影。然而，这些uncouplers（如2,3 - 丁二酮肟）可能有显著的电生理的副作用。 Blebbistatin被证明有23在正常心脏的心脏电生理无不良副作用，因此是一个有前途的解偶联剂光映射。应该指出的是，加速水肿，由于取消收缩也可能影响电。

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>氯化钠</td><td>费舍尔科学，公平的草坪，新泽西州</td><td> S271 - 1 </td></tr><tr><td>氯化钙2（H 2 O） </td><td>费舍尔科学，公平的草坪，新泽西州</td><td> C79 - 500 </td></tr><tr><td>氯化钾</td><td>费舍尔科学，公平的草坪，新泽西州</td><td> S217 - 500 </td></tr><tr><td>氯化镁2（H 2 O） </td><td>费舍尔科学，公平的草坪，新泽西州</td><td> M33 - 500 </td></tr><tr><td>的NaH 2 PO 4（H 2 O） </td><td>费舍尔科学，公平的草坪，新泽西州</td><td> S369 - 500 </td></tr><tr><td>碳酸氢钠3 </td><td>费舍尔科学，公平的草坪，新泽西州</td><td> S233 - 3 </td></tr><tr><td> D -葡萄糖</td><td>费舍尔科学，公平的草坪，新泽西州</td><td> D16 - 1 </td></tr><tr><td> Blebbistatin </td><td> Tocris生物科学，Ellisville，莫</td><td> 1760 </td></tr><tr><td> DI - 4 - ANEPPS </td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> D1199 </td></tr><tr><td> RH237 </td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> S1109 </td></tr><tr><td> Rhod - 2AM </td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> R1244 </td></tr><tr><td>聚醚F127 </td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> P3000MP </td></tr><tr><td>二甲基亚砜（DMSO） </td><td> Sigma公司，圣路易斯，密苏里州</td><td> D2650 </td></tr></tbody></table>

multiparametric optical mapping of the langendorff-perfused rabbit heart

光学成像和荧光探针在1其他的方法，已经不能完成方式在心脏电生理领域的显著先进的研究方法。光的映射允许使用的钙和电压敏感染料，没有身体接触与组织跨膜动作电位和钙瞬变的高空间分辨率的测量。这使得许多条件下使用电极不方便或不可能1心电活动的测量成为可能。例如，光学记录在刺激和除颤后立即提供准确的形态学改变膜电位，而传统的电极技术刺激诱 ​​发的文物遭受刺激，由于电极极化1期间和之后。 的Langendorff灌流兔心脏是人类心脏的生理和病理生理研究最多的车型之一。观察临床心律失常的种类很多，可以在兔心脏模型扼要。结果表明，在兔心脏室性心律失常的波浪纹，有效的心脏和折返波长的大小决定的，在人的心脏2非常相似。它也表明，关键方面对家兔心肌兴奋收缩（欧共体）耦合，如肌浆网（SR）的相对贡献，是非常类似人类EC耦合​​3。在这里，我们目前光的映射实验的基本程序，包括Langendorff灌流系统设置，光学测绘系统设置，隔离和插管，心脏，灌注和染料染色的心脏，兴奋收缩的Langendorff灌注兔心解偶联，并收集光信号。从其他物种比兔与流速的调整，光学，解决方案等，这些方法也可以应用到心脏两个光学测绘系统，进行了阐述。全景测绘系统是用来映射整个兔心脏外膜4-7。该系统提供了一个全球视野，在折返性心律失常和除颤电路的演变，并已用于研究心律失常和抗心律失常治疗 8,9的机制。双绘图系统是用来绘制动作电位（AP）和钙瞬变（CAT），同时从同一领域的观点 10-13 。电交替和诱导心律失常14日至16日，这种做法增强了我们对钙的重要作用的认识。

Watch this Scientific Journal Video about Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart at JoVE.com

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

本文介绍采用全景成像系统中的Langendorff灌注兔心脏进行光学测绘实验的基本程序，和双（电压和钙）的成像方式。

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

multiparametric-optical-mapping-langendorff-perfused-rabbit

Research

JoVE Journal

Bioengineering

22.4K 次观看.  Washington University in St. Louis. 本文介绍采用全景成像系统中的Langendorff灌注兔心脏进行光学测绘实验的基本程序，和双（电压和钙）的成像方式。

视频: Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

科研

生物工程

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

在体外合成的原住民，纤维长间距“和”分段长间距胶原蛋白

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

病人的具体建模之心：的心室纤维方向估计

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

导管内送货到兔乳腺

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

房室心脏瓣膜的双轴力学特性

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...