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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>0.2um 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&#160;</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&#34; OD:3/32&#34; Wall 1/32&#34;</td>
			<td>Component Supply</td>
			<td>TET-031A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing ID:3/32&#34; OD:5/32&#34; Wall 1/32&#34;</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>60mL 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 567nm</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&#160;</td>
			<td>ThorLabs</td>
			<td>FGL590</td>
			<td></td>
		</tr>
		<tr>
			<td>Green Nd-YAG laser 532nm, 500mW</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&#181;m NA 0.39</td>
			<td>ThorLabs</td>
			<td>FT200UMT</td>
			<td></td>
		</tr>
		<tr>
			<td>LEDs blue</td>
			<td>Lumileds</td>
			<td colspan="2">L135-B475003500000</td>
		</tr>
		<tr>
			<td>LEDs green</td>
			<td>Lumileds</td>
			<td colspan="2">L135-G525003500000</td>
		</tr>
		<tr>
			<td>Cube beam splitter, non-polarizing</td>
			<td>ThorLabs</td>
			<td>BS007</td>
			<td></td>
		</tr>
		<tr>
			<td>36&#34; Length, Dovetail Optical Rail</td>
			<td>Edmund Optics</td>
			<td>54-402</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5&#34; Width, Dovetail Carrier</td>
			<td>Edmund Optics</td>
			<td>54-404</td>
			<td></td>
		</tr>
		<tr>
			<td>0.75&#34; Travel, micrometer stage</td>
			<td>Edmund Optics</td>
			<td>37-983</td>
			<td></td>
		</tr>
		<tr>
			<td>Dovetail optical rail 3&#34;</td>
			<td>ThorLabs</td>
			<td>RLA075/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dovetail rail carrier 1&#34;</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&#160;</td>
			<td>ThorLabs</td>
			<td>ST1XY-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum breadboard 6&#34;x6&#34;</td>
			<td>ThorLabs</td>
			<td>MB6</td>
			<td></td>
		</tr>
		<tr>
			<td>Nexus otpical table 4&#39;x6&#39;</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&#160; 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&#160;</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, 25mmx75mmx10mm</td>
			<td>ThorLabs</td>
			<td>BA1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire shelve posts 36&#34;</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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

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, ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

基于3D 荧光显微镜的病毒转录工厂功能成像

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...