Name: local field fluorescence microscopy: imaging cellular signals in intact hearts
Uploaded: 2017-03-08T13:00:00
Description: Watch this Scientific Journal Video about Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts at JoVE.com

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，...

<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 ...

Research

JoVE Journal

Biology

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

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

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

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

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

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

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

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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

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

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

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

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, ...