Name: in vivo calcium imaging in c. elegans body wall muscles
Uploaded: 2019-10-20T12:30:00
Description: Watch this Scientific Journal Video about In Vivo Calcium Imaging in C. elegans Body Wall Muscles at JoVE.com

作者感谢亚历山大·戈特沙尔克博士的ZX1659，含有蠕虫菌株的RCaMP和通道多普辛，金红金博士的slo-1（eg142）蠕虫菌株，以及sca-1（tm5339）蠕虫菌株的国家生物资源项目。

1. 显微镜设置
<ol><li>使用具有荧光功能的复合显微镜。在这项研究中，数据是在装有LED激发的直立显微镜（材料表）上收集的.</li>
	<li>为了正确可视化身体壁肌的荧光变化，使用高放大率目标。<ol>
<li>对于解剖制剂，使用 60x NA 1.0 水浸物（材料表）。</li>
		<li>对于使用纳米珠的制剂，使用 60x NA 1.4 油浸物（材料表）。这种放大倍率可确保足够的肌肉解析到?...

<ol>
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GEC是C.elegans神经生物学的有力工具。以前的工作已经利用钙成像技术检查神经元和肌肉细胞的各种功能，包括感觉和行为反应，具有不同的刺激方法。一些研究已经使用化学刺激触发钙瞬变在感觉ASH神经元22，23或诱导钙波在咽部肌肉24。另一组利用机械刺激，而蠕虫则被关在微流体芯片中，以检查触摸受体神经元?...

本文介绍了利用光遗传学神经元刺激对C.elegans身体壁肌进行体内钙成像的方法。将肌肉表达的遗传编码钙指标（GECI）与蓝光触发神经元去极化，并提供一个系统来清楚地观察诱发的午贴钙瞬变。这避免了使用电刺激，允许对影响午睡钙动力学的突变体进行非侵入性分析。
单荧光基质GECI，如GCaMP，使用单个荧光蛋白分子融合到M13域的肌苷光链激酶在其N端和平静素（CaM）在C端。在钙结合后，对钙具有高亲和力的CaM域会经历一种构象变化，导致荧光蛋白随后的构象变化，导致荧光强度增加1。GCaMP荧光在488nm时激发，因此不适合与通道性荧光结合使用，其激励波长相似，为473nm。因此，为了将钙测量与通道性多普司辛刺激结合使用，GCaMP的绿色荧光蛋白需要替换为红色荧光蛋白mRuby（RCaMP）。使用肌肉表达的RCaMP，结合胆碱能运动神经元表达的渠道多普辛，允许在蠕虫神经肌肉结（NMJ）的研究与同时使用光遗传学和功能成像在同一动物2.
使用通道性多普辛绕过了电刺激的需要，以去极化C.elegans的神经肌肉结，这只能在解剖制剂中实现，从而使这种技术更容易使用和更精确瞄准特定组织时。例如，通道性多普辛以前曾用于C.elegans可逆地激活特定神经元，导致兴奋性神经元或抑制神经元的强健激活3，4。使用光刺激的去极化也绕开神经元损伤的问题，由于直接电刺激。这提供了一个机会来检查许多不同的刺激方案的影响，包括持续和重复的刺激，对后午睡钙动力学4。
C. elegans的透明特性使其成为荧光成像功能分析的理想选择。然而，当刺激NMJ的兴奋性乙酰胆碱神经元时，动物会立即对肌肉收缩4做出反应，使蠕虫的固定性在可视化离散钙的变化时变得至关重要。传统上，药理剂被用来使动物瘫痪。其中一种使用的药物是左旋苯酚，一种胆碱能乙酰胆碱受体激动剂5，6，7。由于左旋基导致兴奋性肌肉受体亚型的持续激活，这种试剂不适合研究肌肉钙动力学。左旋面的作用诱导后脱极化，提升细胞钙，并在登步前刺激后进行闭塞观察。为了避免使用麻痹药物，我们研究了两种替代方法来固定C.elegans。动物要么被粘住，然后解剖打开，露出身体壁的肌肉，类似于现有的C.elegansNMJ电生理学方法8，或纳米珠被用来固定完整的动物9。这两个程序允许可重复的测量休息和唤起肌肉钙瞬变，很容易量化。
本文的方法可用于测量C.elegans中细胞后肝肌肉细胞的基线细胞钙水平和瞬变。给出了采用两种不同固定技术的数据实例。这两种技术都利用光遗传学来消除肌肉细胞的极化，而无需使用电刺激。这些例子证明了这种方法在评估影响蠕虫中贴菌后钙处理的突变的可行性，并指出两种固定方法的优缺点。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>all-trans retinal</td>
			<td>Sigma-Aldrich</td>
			<td>R2500</td>
			<td>Necessary for excitation of channel rhodopsin</td>
		</tr>
		<tr>
			<td>Amber LED</td>
			<td></td>
			<td></td>
			<td>RCaMP illumination</td>
		</tr>
		<tr>
			<td>Arduino UNO</td>
			<td>Mouser</td>
			<td>782-A000066</td>
			<td>Controls fluorescence illumination</td>
		</tr>
		<tr>
			<td>Blue LED</td>
			<td></td>
			<td></td>
			<td>channelrhodopsin illumination</td>
		</tr>
		<tr>
			<td>BX51WI microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Fixed state compound microscope</td>
		</tr>
		<tr>
			<td>Current controlled low noise linear power supply</td>
			<td>Ametek</td>
			<td>Sorenson</td>
			<td>Controls LED intensity</td>
		</tr>
		<tr>
			<td>Igor Pro</td>
			<td>Wavemetrics</td>
			<td>Wavemetrics.com</td>
			<td>Graphing software</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>imagej.nih.gov</td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>LUMFLN 60x water NA 1.4</td>
			<td>Olympus</td>
			<td></td>
			<td>Water immersion objective for dissected preparation</td>
		</tr>
		<tr>
			<td>Master-8 Stimulator</td>
			<td>A.M.P.I</td>
			<td></td>
			<td>Master timer for image acquisition and LED illumination</td>
		</tr>
		<tr>
			<td>Micro-Manager</td>
			<td></td>
			<td>micro-manager.org</td>
			<td>Controls camera acquisition and LED excitiation</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>pco.edge 4.2 CMOS camera</td>
			<td>pco.</td>
			<td>4.2</td>
			<td>High-speed camera</td>
		</tr>
		<tr>
			<td>PlanApo N 60x oil NA 1.4</td>
			<td>Olympus</td>
			<td></td>
			<td>Oil immersion objective for nanobead preparation</td>
		</tr>
		<tr>
			<td>Polybead microspheres</td>
			<td>Polysciences, Inc.</td>
			<td>00876-15</td>
			<td>For worm immobilization</td>
		</tr>
		<tr>
			<td>solid state switches</td>
			<td>Sensata Technologies</td>
			<td>Crydom CMX100D6</td>
			<td>Controls timing of LED illumination</td>
		</tr>
		<tr>
			<td>Transgenic strain, sca-1(tm5339); [zxIs6{Punc17::chop-2 
			(h134R)::yfp,lin-15(+)}; 
			Pmyo3::RCaMP35]</td>
			<td>Richmond Lab</td>
			<td>SY1627</td>
			<td>Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with SERCA mutant allele</td>
		</tr>
		<tr>
			<td>Transgenic strain, slo-1 (eg142); [zxIs6{Punc17::chop-2 
			(h134R)::yfp,lin-15(+)}; 
			Pmyo3::RCaMP35]</td>
			<td>Richmond Lab</td>
			<td></td>
			<td>Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with calcium-activated BK potassium channel mutant allele</td>
		</tr>
		<tr>
			<td>Transgeneic strain, [zxIs6{Punc17::chop-2 
			(h134R)::yfp,lin-15(+)}; 
			Pmyo3::RCaMP35]</td>
			<td>Gottschalk Lab</td>
			<td>ZX1659</td>
			<td>Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line</td>
		</tr>
	</tbody>
</table>

in vivo calcium imaging in c. elegans body wall muscles

模型有机体C. elegans提供了一个出色的系统，以执行体内钙成像。其透明的身体和遗传的可操作性允许基因编码钙传感器的靶向表达。该协议概述了使用这些传感器在靶细胞中钙动力学的体内成像，特别是蠕虫的身体壁肌肉。通过使用前突触通道性多普辛的共同表达，利用蓝光脉冲诱导兴奋运动神经元的乙酰胆碱释放，导致肌肉去极化和细胞质钙的可重复变化水平。讨论了两种蠕虫固定技术，难度不同。这些技术的比较表明，这两种方法都保留了神经肌肉结的生理，并允许钙瞬变的可重现定量。通过配对光遗传学和功能钙成像，可以在各种突变背景中评估午后钙处理和平衡的变化。提供的数据验证了固定技术，并具体考察了C.elegans sco（内科）质状性视网膜钙ATPase和钙激活的BK钾通道在体壁肌肉钙调节中的作用。

这项技术评估了被认为会影响钙处理或肌肉脱极化的突变体的变化。基线荧光水平和荧光瞬变被可视化，并评估肌肉内的细胞钙和钙动力学。重要的是，动物在全视网膜上生长至少三天，以确保视网膜的成功结合，从而随后激活通道性多普司林（图2A）。如果动物没有暴露于全视网膜，肌肉钙瞬时触发（图2B</...

Watch this Scientific Journal Video about In Vivo Calcium Imaging in C. elegans Body Wall Muscles at JoVE.com

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

这种方法提供了一种方法，将光遗传学和基因编码的钙传感器相结合，以成像基线细胞钙水平，以及模型生物体C.elegans身体壁肌肉中诱发的钙瞬变的变化。

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

该协议提供了两种方法，以动员C.Elegans进行体内钙成像的身体壁肌肉。这种方法可以帮助回答与钙平衡和钙处理有关的问题，以及基因可操作生物体中可访问的突触。该技术可以更好地了解调节肌肉钙平衡的机制，因此，在确定通过钙循环调节影响激发收缩耦合的条件或分子通路方面可以保持重要。这些方法可以提供对研究领域的洞察，包括但不限于神经生物学、发育生物学和细胞生物学。这些技术可以适应研究各种细胞类型和C.elegans，其他相关的线虫以及丝虫幼虫。这种方法的视觉演示至关重要，因此实验者能够了解解剖技术，并能够准确评估来自正确固定动物的钙瞬变。首先设置荧光成像显微镜。使用能够以100赫兹进行全帧成像的科学CMOS摄像机，以跟踪钙含量的快速变化。使用在 ImageJ 中运行的微管理器软件控制摄像机采集和 LED 荧光激发。使用连接到外部开源微控制器板的开源电子平台插件来管理控制荧光激动的外部定时脉冲。要控制计时逻辑，请根据制造商的说明在软件中激活微经理采集协议。使用两个 LED 以蓝光刺激通道罗多普辛并记录 RCaMP 变化。使用 am LED 激活通道 rhodopsin，峰值入场波长为 470 纳米和带通滤波器，使用峰值入场波长为 594 纳米的 LED 和带通滤波器激发 RCaMP。使用二色光束组合器共同照亮两个 LED 并将光传输到同一条光路。根据手稿方向，使用 TTL 信号控制的固态开关控制 LED 照明的时序，并设置 LED 强度与当前受控负载噪声线性电源，然后将蓝光仿真协议编程到模拟器中。在此实验中，在仅捕获 RCaMP 荧光两秒钟后打开蓝光模拟，并使用 5 毫秒、2 毫秒的蓝光脉冲（间隔 50 毫秒）来激活通道罗多普辛。如果使用解剖制剂，在低光下执行C.elegans解剖。将动物放在解剖盘中，用硅胶涂层的盖玻片底座，该底座上填充了一毫升钙外细胞溶液，根据手稿指示准备。使用液体局部皮肤粘合剂与蓝色颜色沿蠕虫的后侧粘合动物。并使用玻璃针沿着胶水和蠕虫界面做一个横向角质切口。使用口腔移液器清除蠕虫腔内内脏，然后粘合动物的角质皮瓣，露出腹腔内侧体壁肌肉进行成像。如果使用纳米珠制剂，使用蒸馏水制造熔融 5% Agra 溶液，最终体积为 100 毫升。使用巴斯德移液器将一滴熔融-Agra 溶液放在玻璃滑梯上，并立即将第二个玻璃滑轨放在顶部，使用轻轻的压力创建均匀的 Agra 垫。将顶部幻灯片和大约四个微升聚苯乙烯纳米珠到垫中间。在低光下，在纳米珠溶液中加入四到六个C.elegans，确保动物不会彼此躺在上面，并小心地在上面放置一个盖玻片。准备好成像时，将准备好的幻灯片或解剖盘放在显微镜上，并使用 10 倍的放大倍率和昏暗的明亮场照明找到并聚焦在蠕虫上。在 RCaMP 荧光激发中切换到 60 倍放大，以识别外阴前部和正确的声平面中内侧体壁肌肉。然后，通过拉出切换并单击数据采集软件中的实时图像路径，将图像路径从目镜更改为相机。单击数据采集软件中的 ORI 按钮，围绕正在关注的肌肉创建一个框。在刺激器上，打开先前编程的蓝光刺激路径，然后单击"获取"成像软件以捕获图像。当C.elegans生长在全跨视网膜上，成功结合视网膜并激活通道罗多普辛时，可以唤起钙的瞬态。如果动物没有暴露在全跨视网膜没有肌肉钙瞬态被触发。该协议用于研究功能 sca-1 突变体的损失，这些突变体影响由 sca-1 基因编码的沙原体性钙泵。但是，与对照组相比，在突变体中观察到RCaMP荧光基线水平的解剖制剂增加，这表明突变需要休息细胞质钙水平升高。与对照体相比，sca-1突变体的峰值钙含量显著降低。然而，在上升的高峰时间或半衰减时间没有变化。重要的是，使用技术上挑战性较低的纳米珠制剂可以观察到类似的结果。评估了破坏钙激活大钾通道的功能慢突变体损失，以调查间接影响C.elegans钙处理的突变体。当测量RCaMP的基线水平时，与对控组相比，突变体显示的荧光增加。在评估钙瞬态动力学时，未看到峰值钙水平的变化。然而，slo-1（如142）突变体在峰值时间的上升显著增加。完成手术时要记住的最重要的一步是准备全跨视网膜的实验动物。如果没有此步骤，通道红松素将处于非活动状态，防止引发光诱导钙瞬变。按照这个程序，电生理学和行为分析可以进行评估任何观察到的钙平衡变化的功能影响。使用此处概述的固定化方法，为进一步探索钙动力学铺平了道路，并证明可以使用远没有挑战性的珠固定技术对光遗传学神经元刺激和肌肉钙瞬变捕获进行类似结果的观察。

Research

JoVE Journal

Biochemistry

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

结合拉曼成像和多变量分析可视化植物细胞壁中的木质素，纤维素和半纤维素

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

植物甘露聚糖细胞壁多糖的结构表征

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

A Colorimetric Method for Measuring Iron Content in Plants

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

蜜蜂匀浆中磷脂酶 C 活性的检测

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

使用荧光寿命成像在老化C.Elegans中淀粉样蛋白结构的表征

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...