我们感谢艾丽西亚Mattiazzi博士所提出的工作提出了批评讨论。这项工作是由美国国立卫生研究院从赠款（R01 HL-084487），以支持ALE。

该协议并且所有小鼠处理批准了加州大学默塞德机构动物护理和使用委员会（编号2008-201）。与鹦鹉实验于1999年根据由委内瑞拉科学研究所（IVIC）的科学委员会设立动物使用一般政策进行的。 1.的Langendorff成立准备<ol><li>制备的台罗德氏溶液含有以mM以下溶质浓度:140的NaCl，5.4氯化钾，2的CaCl 2，1 MgCl 2的，0.33 的 Na 2 HPO 4，10葡萄糖，10 HEPES。调整台罗德氏溶液，以7.4用NaOH的pH值和过滤通过0.22微米的过滤器将溶液。 </li><li>装载台氏溶液到60毫升的注射器和设置在图4所示11，12中的水平的Langendorff的所有管道。确保消除所有气泡。 </li><li><stro纳克&gt;下平衡的Tyrode溶液用100％O 2的用浸没塑料"空气石"，如图4所示。 <ol><li>塑料管连接到O 2罐。 </li><li>添加塑料底座适配器降低5"管插入的60毫升注射器台氏液。 </li><li>塑料气石连接来了5"管的末端所以O 2将泡出到台氏液。 </li></ol></li><li>放置一个非吸收性外科缝线围绕用作套管针。针被连接到歧管（ 见图4），允许复古灌注不同的解决方案。最后，心脏的主动脉将空心进针。 </li></ol> 2.动物的制备与心脏解剖<ol><li>肝素称量并注入鼠标（例如，鼠标的重量20克，用20单位或200微升注入）颈椎脱臼安乐死前15分钟。麻醉鹦鹉根据:Ø您的IACUC协议的动物使用教程，然后继续进行颈椎脱位。 注:使用8周龄小鼠或20克鹦鹉。 </li><li> 从安乐死后胸腔中取出心脏。作为描述小鼠中提取完全相同的方式鹦鹉心。 <ol><li>清洁用乙醇鼠标胸口。 </li><li>使用夹层剪刀，做一个切口下腹部，然后切开两侧朝脖子。 </li><li>拉回切割组织和引脚下来。 </li><li>切隔膜。切割膜片时要避免损坏心脏要小心。 </li><li>取出肺和周围的组织。 </li><li>使用镊子舀心脏不受挤压它。尽可能长时间地切主动脉。 </li></ol></li><li>与约1毫升台氏液运送一个小权衡船的心脏。 </li><li>使用非吸收性外科缝线，通过扎到大动脉水平的Langendorff设备针。领带的心脏主动脉有两个细镊子的帮助。打开位于与含台氏液60毫升注射器系列阀门开始复古灌注。 </li><li>让心脏稳定10分钟。利用这段时间来清洁血液和加载染料之前心脏周围的脂肪组织。拉用镊子心脏的基地附近的脂肪组织，并用小解剖剪刀剪。一定要做到这一点解剖显微镜的视野下。 </li></ol> 3.胞质钙测量:准备染料RHOD-2AM <ol><li>添加20％普朗尼克20μL的（非离子表面活性剂）在DMSO中，通过在含有该染料的50微克的染料制造商提供的特殊包装的塑料小瓶中。 </li><li>吹打向上和向下，避免气泡混合。 </li><li>与非离子表面活性剂和从特殊包装的塑料管形瓶中的染料成透明玻璃小瓶转移混合DMSO中。加入1毫升台氏SOLU的重刑透明玻璃小瓶。 </li><li>超声处理用于在槽式声波处理器15-20分钟。 </li><li> 使用蠕动泵在室温下30分钟灌注染料。 <ol><li>放置染料在染料腔室。 </li><li>使用机械夹具压缩所有连接到歧管的另一管道线。夹具被放置在歧管的上方。这将防止任何回流到连接到60毫升的注射器管。 </li><li>打开灌注蠕动泵开始循环的染料。立即关闭60毫升注射器低于三通阀。 </li><li>定位连接到旁边的心脏抽吸蠕动泵再循环已灌注到心脏的染料的小管。 </li></ol></li></ol> 4.内部-SR 钙测量:准备染料幅度- Fluo4AM <ol><li>以相同的方式作为RHOD-2AM制备幅度 - Fluo4AM。请参见3逐步说明。 </li><li> AFTER装入染料，打开位于含台氏液，开始复古灌注的60毫升注射器系列阀门。一定要去掉上面歧管钳。 </li><li>加台罗德氏溶液，以在水平腔和温热至37℃。 </li><li>复古与灌流45分钟台氏液以去除细胞内的染料。 </li></ol> 5.膜电位测量:准备染料迪8 ANEPPS <ol><li>加入5毫升99％的乙醇对包含5毫克染料的染料小瓶中。 </li><li>分装10μL到使用中继器吸管500个人的1毫升塑料小瓶。 </li><li>变干的速度真空并储存在-20℃。 </li><li>添加20μL的20％泊洛尼克于DMSO至塑料小瓶中的干燥的染料的10微克。 </li><li>吹打向上和向下，避免气泡混合。 </li><li>含DMSO加普朗尼克和染料混合传输从塑料小瓶量筒（10毫升）中。加台氏溶液至最终沃5毫升lume明。 </li><li>超声处理用于在槽式声波处理器20-25分钟。 </li><li>灌注到心脏，使用蠕动泵30分钟。请参阅逐步说明3.5节。 </li></ol> 6。心外膜录音信号<ol><li>装载染料后，通过去除所述歧管上方的钳复古灌注与台罗德氏溶液的心脏。打开位于与含有台罗德氏溶液的60毫升注射器系列阀门。 10分钟复古灌流台氏液，以稳定心脏。 </li><li>填充台氏溶液的水平腔和转动珀尔帖单元上以使浴温至37℃。 </li><li> 心脏的表面上的光纤位置。 <ol><li>放置一个2毫升吸管内的光纤，然后将吸管连接到一个显微。 </li><li>使用显微稍微按下光纤对LV的表面上。 </li></ol></li><li> 外部的步伐老天由波发生器控制的刺激室温。 <ol><li>程序中的波发生器提供方波脉冲为1ms的宽度。 </li><li>设置为外部同步刺激器和外部输入连接到波发生器。 </li><li>为了刺激器的每个输出，并在最后的焊接针针连接线。 </li><li>放置两个针灸针在心脏约3mm彼此分开的顶点。 </li><li>针被放置在组织后才，打开刺激器的输出，以防止触电。 </li></ol></li><li>在采集软件，调整频率采集到10kHz。 </li></ol> 7。记录心内膜信号<ol><li>参照步骤6.1和6.2加载染料后，以稳定的心脏。 </li><li>关于使用光纤的规模急剧23Ga点，使心脏的表面的小孔中隔附近的LV。 </li> &lt;li&gt;将一玻璃体内手术巩膜适配器在使用显微第一光纤定位到心内膜，以帮助。 </li><li>外部踱步的心脏。参见步骤6.4。 </li><li>在采集软件，调整频率采集到10kHz。 </li></ol>

<ol>
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本文在描述局部场荧光技术来评估心肌细胞体外的功能中心。这些细胞在耦合环境的研究不仅更加生理，但也是高度适宜用于评估器官级病状。底层兴奋-收缩偶联（ECC）的蜂窝事件可以在与使用监测细胞内Ca 2+动力学分子探针的整个器官水平进行评估（RHOD 2细胞内的Ca 2+，幅度- Fluo4 SR的Ca 2+）和电活动（二 - 8-ANEPPS）。这里，我们已经提出了三种LFFM萤光技术，既激?...

心脏是心血管系统的中央机关。心脏的收缩是由细胞内增加钙离子浓度开始。在细胞内Ca 2+释放电兴奋和变化之间的关系已经在历史研究了酶促解离的细胞1,2。然而，心脏细胞电，代谢和机械地联接3,4。当隔离，肌细胞不仅是身体上脱开，但是从不同层细胞的解离5中混合。此外，尽管已经从分离细胞的研究中出现电压钳条件下的巨大优势，6，7，8心脏的内在性质为电合胞体ALWAYS构成如何功能不同的是从那些存在于组织3解离的细胞的问题。 在这个手稿中，我们描述了在完整的心脏采用局部场荧光显微镜（LFFM）技术在心脏生理知识获得的进步。 LFFM使用荧光指标来衡量多种生理变量，如细胞内钙离子 ，内肌质网（SR） 钙和膜电位。这些测量可以同时并结合心室压9,10，心电图9，电气动作电位（AP）的，离子电流的录音和笼化合物4的闪光光解，11来获得。此外，这些测量可以通过在更高的频率接近起搏完整心脏得到R以生理率。尽管一些文章9，11，12，13，14已使用LFFM技术我们集团公布，技术与该技术相关的复杂的推定阻止在心脏和其他器官研究体外生理现象它的大规模使用。 所述LFFM技术（ 图1）是基于使用在与组织接触的多模光纤获得萤光测量。像任何接触荧光成像技术，光学分辨率取决于直径和光纤的数值孔径（NA）。光纤的较高NA和较小直径将增加测量的空间分辨率。的NA和纤维直径的范围可以从0.22至0.66，并从50微米至1毫米。在压痕的NA将通过接受从较大的立体角到达光子提高信噪比（S / N）。为了充当萤光设备中，光束被聚焦到光纤用的非球面透镜或落射荧光物镜，其中透镜的NA和光纤匹配。该匹配最大化为激励和用于收集回由荧光团发射的光子的能量传递。 为了激发在组织中加载的外源性荧光指示器，不同的光源和照明模式都可以被利用。我们使用脉冲局部场荧光显微镜3，12（PLFFM）开拓性的研究中采用的低成本皮秒激光器（ 图1a，PLFFM）。这种类型的光源的具有照明区域下荧光团分子的激发大级分的巨大优势，而基本上不漂白由于染料以短脉冲持续时间12。此外，使用超短脉冲的允许的染料12的荧光寿命的评估。荧光寿命是可用于量化绑定到的 Ca 2+染料分子的分数属性。不幸的是，从脉冲到脉冲的脉冲和变化的幅度的时间抖动限制本实验策略来的情况下通过配体结合到染料产生的荧光变化是大的应用程序。 连续波（CW）激光器通常用作LFFM（ 图1b，CLFFM）的主要照明源。激光束可以连续照亮组织或可以ferroelectrically调制。的光束的强电介质调制让光微秒脉冲的产生。这种调制可以通过外部硬件进行控制。这个过程不仅极大地降低了Ť他光脉冲的时间抖动也可以让混合不同波长的光束。束的混合是通过从不同的激光器多路复用射线进行。其结果是，具有不同的光谱特性的多个染料可兴奋来执行的各种生理变量的测量，例如，RHOD-2细胞内的Ca 2+，MagFluo4帧内-SR 的 Ca 2+和二-8- ANEPPS为膜电位。 尽管激光器本各种优点如LFFM光源，其它类型的光源可以使用，包括发光二极管（LED）。在这种情况下，激励光源组成的InGaN LED（ 图1c，PLEDFM）的。在发光二极管中，当从导带的电子与价带中空穴复合光子自发发射。与固态激光器的区别在于，发射不被其他光子激发。这导致了非相干光束和LED的更宽的光谱发射。 可用于不同类型的高功率LED。对于AP的记录用迪-8- ANEPPS和钙使用的Fluo-4或幅度-的Fluo-4，我们使用了在485纳米（蓝）和20nm的半值宽度具有典型的峰值发射的LED记录2+瞬变（ 图1d）。对于记录有RHOD-2 的 Ca 2+瞬变，该LED在540nm（绿色）和35纳米（ 图1d）的半高宽了一个典型的峰值发射。 LED发出的波长频段，因此需要过滤器来缩小他们的光谱发射。另外，可在1.6千赫为20微秒的持续时间的比率来产生脉冲光。这些LED用一个快速的功率MOSFET场效应晶体管脉冲。同时进行的录像用不同的指标可通过时分复用的指示灯来执行。不幸的是，由LED发射的光更难以集中到光纤相比的激光束。因此，我们的主要缺点ING的LED是，它们的发射轮廓具有从主轴线的角位移（±15°），和一个辅助光学必须使用纠正它。 在所有先前描述的光学结构中，激发光被反射用的分色镜的帮助。光束随后通过非球面透镜和一个显微镜物镜上被定位在组织中的多模光纤聚焦。正如在任何萤光布置中，分色镜还用于激励从所发射的光中分离出来。所发出的光频谱旅行回到通过屏障过滤器，以消除任何反射的激发。最后，发射的光与目标聚焦到光检测器（ 图1）。 从光到电的电流的传导是由硅雪崩光电二极管进行。这些二极管具有快速响应和高灵敏度，允许低光检测。该由雪崩光电二极管产生的光电流可以用两种方法来扩增:具有跨阻抗放大器的电阻反馈元件（ 图1E）或通过积分将电流转换成电压（ 图1F）。使用第一种方式，输出电压正比于光电流和反馈电阻。电阻检测皮秒激光脉冲的一个典型的例子示于图2a，2b和2c。面板2a示出了跨阻放大器的输出和面板2b示出了具有一个星号（*）指示的时间间隔的一个时间扩展。峰值跟踪算法被实施以检测荧光反应12峰（红色）和底座（绿色）。碱荧光的测量提供了受环境韧带引入两个雪崩光电二极管的暗电流和干扰的信息HT和电磁耦合。峰和碱的表示示于图2c中。该图中示出了由打浆鹦鹉心脏的心动周期中结合到钙染料（RHOD-2）发出的荧光。 在第二种方法中，积分器的输出电压是电流和电容反馈的函数（ 图2d，2e和2f）的 。 图2F示出了两个连续的积分周期:第一，没有外部照明和所述第二带从一个脉冲的LED施加的光脉冲。的详细描述示于图2g和2小时 。这种方法，虽然更费力，提供了一个较大的S / N由于没有在反馈电容器热噪声。仪器包括产生激发光的所有的控制和多路复用并命令探头整合和水库的定时阶段等周期。这通常是用一个数字信号处理电路，还通过计算数据的一个在线回归进行综合输出信号的数字分化进行。在使用电阻反馈的情况下，可使用任何A / D采集板。 最后，我们的LFFM技术是高度通用的，并且可以适合于从一个以上的区域进行记录。在光路中加入一光束分离器可以使我们的光分成两个光纤。然后各光纤可以被放置在一个组织，以独立地，激发并从外源荧光探针记录发射的不同区域。这一修改使我们能够评估区域如何解剖学上的差异影响生理变量。 图3示出一个分束器所采用分裂，使得两个光纤被用来测量透电或细胞内的CW激发光的[Ca 2+]的水平机智^ h轻微的侵入性。透的信号可以通过将在内膜一种纤维和其它心室壁的外膜层上进行记录。因此，LFFM技术具有以测量蜂窝信号的不同区域的时间过程的能力，并且可以使用，如果在病理情况下发生的区域的变化进行测试。

<table><tbody><tr><td>Sodium chloride</td><td>Sigma</td><td>7647-14-5</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Sigma</td><td>50-99-7</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>7447-40-7</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td></td></tr><tr><td>Sodium phosphate</td><td>Sigma</td><td>10049-21-5</td><td></td></tr><tr><td>Calcium chloride solution</td><td>Sigma</td><td>10043-52-4</td><td></td></tr><tr><td>Magnesium chloride solution</td><td>Sigma</td><td>7786-30-3</td><td></td></tr><tr><td>Sodium hyrdoxide</td><td>Sigma</td><td>S-8045</td><td></td></tr><tr><td>0.2 &amp;mu;m nylon membrane filter</td><td>Whatman</td><td>7402-004</td><td></td></tr><tr><td>Manifold MPP</td><td>Warner</td><td>64-0216&amp;nbsp;</td><td></td></tr><tr><td>21G1.5 Precision glide needle</td><td>B-D</td><td>305167</td><td></td></tr><tr><td>Black Braided silk string (non-absorbable surgical suture)</td><td>AllMech Tech</td><td>LOOK-SP105</td><td></td></tr><tr><td>Heparin sodium injection 1000USP/mL</td><td>AllMech Tech</td><td>NDC63323-540-11</td><td></td></tr><tr><td>DMSO D8779</td><td>Sigma</td><td>67-68-5</td><td></td></tr><tr><td>Blebbistatin</td><td>Sigma</td><td>856925-71-8</td><td></td></tr><tr><td>Pluronic F-127 20% solution</td><td>Biotium</td><td>59004</td><td></td></tr><tr><td>Materflex C/L peristaltic pump</td><td>Cole-Parmer</td><td>77122-26</td><td></td></tr><tr><td>Isostim stimulator</td><td>World Precision Instruments</td><td>A320RC</td><td></td></tr><tr><td>Waveform generator</td><td>Teledyne Lecroy</td><td>WaveStation 2012</td><td></td></tr><tr><td>Ultrasonic cleaner FS20</td><td>Fisher Scientific</td><td>1533530</td><td></td></tr><tr><td>Tygon tubing ID:1/32&quot; OD:3/32&quot; Wall 1/32&quot;</td><td>Component Supply</td><td>TET-031A</td><td></td></tr><tr><td>Tygon tubing ID:3/32&quot; OD:5/32&quot; Wall 1/32&quot;</td><td>Component Supply</td><td>TET-094A</td><td></td></tr><tr><td>Adapter luer lock to 3-way valve</td><td>Cole-Parmer</td><td>EW-31200-80</td><td></td></tr><tr><td>Tee adapters and plastic fittings</td><td>Cole-Parmer</td><td>6365-90</td><td></td></tr><tr><td>Plastic clamp</td><td>WaterZoo</td><td>2465</td><td></td></tr><tr><td>Peltier</td><td>TE Technology</td><td>TE-127-2.0-2.5</td><td></td></tr><tr><td>Rhod-2AM</td><td>ThermoFisher Scientific</td><td>R1245MP</td><td></td></tr><tr><td>Di-8-ANEPPS</td><td>ThermoFisher Scientific</td><td>D3167</td><td></td></tr><tr><td>Mag-Fluo-4AM</td><td>ThermoFisher Scientific</td><td>M14206</td><td></td></tr><tr><td>Acupunture needles</td><td>LHASA</td><td>TC1.20x13</td><td></td></tr><tr><td>60 mL BD syringe with luer-lok</td><td>Fisher Scientific</td><td>14-820-11</td><td></td></tr><tr><td>LabView</td><td>National Instruments</td><td></td><td></td></tr><tr><td>Speed vacuum Eppendorf Vagufuge</td><td>Fisher Scientific</td><td>07-748-13</td><td></td></tr><tr><td>Digital signal processing circuit DSP TMS 320</td><td>Texas Instrument</td><td></td><td></td></tr><tr><td>Longpass Dichroic mirror 567 nm</td><td>ThorLabs</td><td>DMLP567L</td><td></td></tr><tr><td>Objective 10X NA 0.25 DIN AchromaticFinite Intl Standard Objective</td><td>Edmund Optics</td><td>Stock# 33-437</td><td></td></tr><tr><td>Objective 20X NA 0.40 DIN Achromatic Finite Intl Standard Objective</td><td>Edmund Optics</td><td>Stock# 33-438</td><td></td></tr><tr><td>Longpass colored glass filter 590 nm&amp;nbsp;</td><td>ThorLabs</td><td>FGL590</td><td></td></tr><tr><td>Green Nd-YAG laser 532 nm, 500 mW</td><td></td><td></td><td></td></tr><tr><td>Micromanipulator for laser</td><td>Siskiyou</td><td>MX130R</td><td></td></tr><tr><td>Multimode fiber optic 200 &amp;micro;m NA 0.39</td><td>ThorLabs</td><td>FT200UMT</td><td></td></tr><tr><td>LEDs blue</td><td>Lumileds</td><td>L135-B475003500000</td><td></td></tr><tr><td>LEDs green</td><td>Lumileds</td><td>L135-G525003500000</td><td></td></tr><tr><td>Cube beam splitter, non-polarizing</td><td>ThorLabs</td><td>BS007</td><td></td></tr><tr><td>36&quot; Length, Dovetail Optical Rail</td><td>Edmund Optics</td><td>54-402</td><td></td></tr><tr><td>2.5&quot; Width, Dovetail Carrier</td><td>Edmund Optics</td><td>54-404</td><td></td></tr><tr><td>0.75&quot; Travel, micrometer stage</td><td>Edmund Optics</td><td>37-983</td><td></td></tr><tr><td>Dovetail optical rail 3&quot;</td><td>ThorLabs</td><td>RLA075/M</td><td></td></tr><tr><td>Dovetail rail carrier 1&quot;</td><td>ThorLabs</td><td>RC1</td><td></td></tr><tr><td>Avalanche photodiode Helix 902</td><td>Digi-Key</td><td>HELIX-902-200</td><td></td></tr><tr><td>Objective holder XY Translator&amp;nbsp;</td><td>ThorLabs</td><td>ST1XY-S</td><td></td></tr><tr><td>Aluminum breadboard 6&quot; x 6&quot;</td><td>ThorLabs</td><td>MB6</td><td></td></tr><tr><td>Nexus otpical table 4&#039; x 6&#039;</td><td>ThorLabs</td><td>T46HK</td><td></td></tr><tr><td>Stainless steel cap screws</td><td>ThorLabs</td><td>HW-KIT5/M</td><td></td></tr><tr><td>Acrylic&amp;nbsp; sheet</td><td>Home Depot/Lowes</td><td></td><td></td></tr><tr><td>Sylgard Silicone elastomer kit</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr><tr><td>Epoxy gel&amp;nbsp;</td><td>Walmart/Home Depot</td><td>2-part 5 min clr 1oz</td><td></td></tr><tr><td>21G1.5 Precision glide needle</td><td>B-D</td><td>305167</td><td></td></tr><tr><td>23G Precision glide needle</td><td>B-D</td><td>305145</td><td></td></tr><tr><td>Mounting base, 25 mm x 75 mm x 10 mm</td><td>ThorLabs</td><td>BA1/M</td><td></td></tr><tr><td>Wire shelve posts 36&quot;</td><td>Alera</td><td>AALESW59PO36SR</td><td></td></tr><tr><td>Wire shelves</td><td>Alera</td><td>ALESW582424SR</td><td></td></tr><tr><td>Post and angle clamp</td><td>ThorLabs</td><td>SWC/M-P5</td><td></td></tr><tr><td>Glass syringe for dye chamber</td><td>Wheaton</td><td>W851020</td><td></td></tr><tr><td>Rubber stopper</td><td>Home Science Tools</td><td>CE-STOP01C</td><td></td></tr></tbody></table>

local field fluorescence microscopy: imaging cellular signals in intact hearts

在心脏，分子信号研究在离体心肌细胞通常执行。然而，许多如局部缺血和心律失常病理情况只能完全在整个器官水平的理解。在这里，我们提出局域场荧光显微镜（LFFM）的光谱技术，允许在完整心脏细胞信号的测量。该技术是基于一个的Langendorff灌注的心脏和光纤，记录荧光信号的组合。 LFFM在心血管生理学领域的各种应用研究正常和病理条件下的心脏。多个心肌变量可以使用不同的荧光指标进行监测。这些措施包括细胞内钙离子浓度 ，内肌浆网内钙和膜电位。外源荧光探针很高兴与LFFM落射荧光技术的三种不同的安排检测发出的荧光■在本文提出。这些技术中的中心差异是用于激励和激励光进行调制的方式的光源的类型。脉冲LFFM（PLFFM）采用激光光脉冲，同时连续波LFFM（CLFFM）使用连续激光激发。最后，发光二极管（LED）被用作第三光源。该非相干安排被称为脉冲LED荧光显微镜（PLEDFM）。

美联社和钙 瞬变的心内膜和心外膜 以横跨心室壁比较信号，一个光纤被定位在内膜和另一个在外膜。从心内膜从心外膜记录与一个AP的形态相比较是评估透功能的最佳方式。室壁是高度异质性，因此，受影响的形态是在这两个区域有很大不同。这是众所周知的，该心内膜少我到...

Watch this Scientific Journal Video about Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts at JoVE.com

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

在心脏，分子事件坐标器官的电气和收缩功能。一套这里提出局域场荧光显微镜技术能使在完整的心中蜂窝变量的记录。识别定义心脏功能的机制是关键了解的心脏在病理情况下是如何工作的。

局部场荧光显微镜:成像在完整的心蜂窝信号

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and ...

local-field-fluorescence-microscopy-imaging-cellular-signals-intact

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.2K 次观看.  University of California, Merced. 在心脏，分子事件坐标器官的电气和收缩功能。一套这里提出局域场荧光显微镜技术能使在完整的心中蜂窝变量的记录。识别定义心脏功能的机制是关键了解的心脏在病理情况下是如何工作的。 

视频: Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

成像清除胚胎和出生后心脏在单细胞分辨率

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

科研

发育生物学

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

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 ...

生物学

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

通过清除心脏三维成像捕获目标细胞群的心脏损伤反应

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

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. ...

生物工程

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

分离的月毛心室肌细胞的光学成像

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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 ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

全小鼠心脏的介观光学成像

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

局部场荧光显微镜:成像在完整的心蜂窝信号

摘要

探索更多视频

此视频中的章节

成像清除胚胎和出生后心脏在单细胞分辨率

光学测绘的Langendorff灌注大鼠心脏

通过清除心脏三维成像捕获目标细胞群的心脏损伤反应

光片荧光显微术在小鼠心脏研究中的应用

分离的月毛心室肌细胞的光学成像

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

成像本地CA

的内肌浆网钙光学标测

全小鼠心脏的介观光学成像

局部场荧光显微镜:成像在完整的心蜂窝信号

摘要

探索更多视频

此视频中的章节

成像清除胚胎和出生后心脏在单细胞分辨率

光学测绘的Langendorff灌注大鼠心脏

通过清除心脏三维成像捕获目标细胞群的心脏损伤反应

光片荧光显微术在小鼠心脏研究中的应用

分离的月毛心室肌细胞的光学成像

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

成像本地CA

的内肌浆网钙光学标测

全小鼠心脏的介观光学成像

同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo