Name: imaging calcium in drosophila at egg activation
Uploaded: 2016-08-06T14:00:00
Duration: 7 min 45 s
Description: Watch this Scientific Journal Video about Imaging Calcium in Drosophila at Egg Activation at JoVE.com

我们感谢劳拉班普顿，亚历克斯·戴维森，理查德·帕顿，阿查里雅田对这个手稿的准备过程中提供援助;马里亚纳Wolfner对鸡蛋激活讨论;马特·韦兰成像的支持;和胡楠在实验室普遍支持。 这项工作是由剑桥，ISSF的大学TTW [授权号097814]的支持。

注：在常温下进行所有步骤，除非另有说明。 1.准备解剖注意：这里描述的步骤都是按照E. Gavis，普林斯顿大学和威尔等。人 18。前成像执行以下步骤两天。 <ol><li>取含有大约10毫升固体食物蝇小瓶中并干燥酵母的少量添加到一个角，小于1g是足够的。 注：有关飞小瓶或食品信息，请参阅“ 果蝇维护”19。 </li><li>加入1 - 水3滴，以水化酵母。 </li><li>预留小瓶在45°角，并允许3 - 为对酵母5分钟以占用水。 </li><li>而酵母是吸收水，选择一个瓶表达由浴盆-VP16GAL4驱动UASt- MYR GCaMP5 20蝇（简称GCaMP5），该最近出现（一myristoy被选择的GCaMP5的迟来形式通过在蛋拴系的GECI向膜），以提供高的信噪比。 </li><li>麻醉苍蝇在瓶的 CO 2气体。一定要保持瓶口倒置，以免失去苍蝇在湿的食物。 </li><li>尖端麻醉苍蝇到CO 2垫和排序10 - 15名女性和男性5使用解剖显微镜下画笔。注：这是标准的，包括男性为解剖提供健康的女性。 </li><li>当来自步骤1.3小瓶准备就绪时，所选择的苍蝇添加到小瓶中。注意保持小瓶水平，直到苍蝇变得活跃。 </li><li>放置在25℃的小瓶约36小时。 </li><li>从CO 2垫到瓶子或丢弃返回剩余的苍蝇。 </li></ol> 2.解剖果蝇卵巢注意：这里描述的步骤都是按照威尔等。人。 <s达&gt; 18。 <ol><li> 36小时后，从CO 2 yeasted小瓶麻醉苍蝇。 </li><li>一旦小瓶内部的苍蝇麻醉提示他们到CO 2的垫用解剖显微镜下漆刷进行排序。 </li><li>选择一个女性。 </li><li>隔离从阴两个卵巢。有关完整详细的协议看威尔等。人 18。综上所述： <ol><li>将含氧卤烃油（系列95）的一小滴在1.5号，22×40mm的盖玻片。 </li><li>把握用镊子阴在腹部的前部。 </li><li>在解剖显微镜下握住女性，使用第二套钳轻轻地去除女性的后路。 </li><li>擦去从第二钳后组织。 </li><li>挤在与由前腹部的后部的第2钳子腹部空腔。这两个不透明卵巢应坚持镊子时，外面的联邦紧急事务管理局勒。 </li><li>触摸卵巢进油。 </li><li>擦去镊子除去从钳子的任何组织。 </li><li>从一飞获得3盖玻片，每个包含卵巢 - 重复上述步骤（2.4.7 2.4.1）。 </li></ol></li></ol> 3.隔离单个成熟卵子<ol><li>根据与来自步骤2.4设置为4X，在第一盖玻片的倍率的标准解剖显微镜，通过撕开输卵管分开的两个卵巢。 </li><li>介绍一对封闭钳成一个卵巢的中心，注意不要刺破任何蛋室。 </li><li>插入一个标准的解剖探入卵巢的中心封闭镊子旁边。 </li><li>通过朝后极，其中，成熟的卵子（阶段14蛋室）位于卵巢轻轻拖曳解剖探针。 </li><li>重复步骤3.1 - 取决于卵巢和蛋的数量的大小的5倍 - 3.4 2。 注：CO 2的添加</子&gt;通常会导致将出现与凸起背侧附属物比预沉积卵较大的单个蛋的沉积。因为它已经被阴内激活这个蛋应该丢弃成像。 注：成熟的卵子有可能轻松地从卵巢结缔组织分隔。 </li><li>如果没有成熟的卵子都是卵巢的外部可见，继续轻轻地用解剖探头戏弄。 </li><li>在盖玻片的中心的线5 - 一旦成熟卵是对卵巢的外盖玻片可见，操纵3。 注：为达到最佳效果，沿鸡蛋的一侧轻轻地运行探头。 注意：不要拉背附肢，否则会损坏卵子。 注意：根据未来成像设置，对准鸡蛋约200微米相距垂直于盖玻片最大成像区。 </li><li>一旦对准，通过使用f起对准鸡蛋拖动距离取出卵巢组织的其余部分orceps。 </li><li>通过与医疗擦拭涂抹角落删除多余的油。小心不要碰到成熟的卵子与医疗擦拭那些接触将失去作为成熟的卵子。 </li><li>上绘制盖玻片的十字准线与一个标记，以简化定位在显微镜下的样品。 </li><li>坐落在一个安全的地方盖玻片，离开盖玻片上的准备鸡蛋室休息5 - 10分钟。这允许蛋在油定居。样品可以使用到1小时后淋巴结清扫术。 </li><li>重复步骤3.1 - 3.11盖玻片的其余部分与他们的卵巢。 </li></ol> 4.准备成像<ol><li>带来制备活化缓冲器15至室温。 注意：激活缓冲液是低渗缓冲液：3.3毫的NaH 2 PO 4，16.6毫KH 2 PO 4，10 mM氯化钠，50mM的氯化钾，5％的聚乙二醇8000，2毫氯化钙2，调至pH 6.4以1： 5比氢氧化钠：KOH。有关详细信息，请参阅马霍瓦尔德，1983 15。活化缓冲液的等分可以储存在-20℃下个月，并在4℃下最多2周。 </li><li>精心准备运盖玻片，活化缓冲液和玻璃吸管的成像设备。 注意：宽视场，共聚焦，旋转光盘或光片显微镜都可以使用。我们建议使用倒置显微镜。利用共聚焦显微镜获得了我们的代表性的结果。 </li><li>适合成像整个成熟的卵子一个目标应（这里：20X 0.7 NA）选择。 </li><li>安装在显微镜阶段的第一个准备盖玻片。小心不要打破舞台上的盖玻片。 </li><li>定性选择的视场与成熟卵室进行激活。不要刺破形象蛋室或那些在细胞膜起泡。 注意：对于步骤4.6 - 4.7，软件命令将视显微镜和制造商的不同而不同。 </li><li>组影像对位米标准GFP激发（488纳米）和发射（500 - 580纳米）。还成立了以可视化和定向的成熟卵子和流离失所油明视场视图。 </li><li>选择最低的Z平面，其中成熟卵是可见的，并设置一个完整的Z堆栈应采取的40微米每步约2微米。所以Z堆栈收购之间没有延迟设置参数。设置的时间序列来捕获约30分钟。 </li></ol> 5.成像离体激活蛋<ol><li>启动图像捕捉。 </li><li>等待1 - 要获得2个全Z-栈。这说明之前激活成熟的卵子。 </li><li>撤回大约300微升活化缓冲剂成玻璃吸管。 </li><li>定位含有活化缓冲液中在45°角盖玻片上方的玻璃吸管的前端，慢慢地吸移管移入光束路径直到它直接上面的成熟卵被成像。玻璃吸管应为1 - 2厘米油上方。 </li><li>从玻璃吸管活化缓冲液2滴，在不到5秒 - 加入1。这应该置换的油。避免加入过量的活化缓冲或触摸与玻璃吸管蛋，因为它可以导致成熟卵子的位移。小心不要碰到盖玻片用吸管，因为这可能会导致在焦平面或气泡的变化，形成了客观和盖玻片之间的浸油。 </li><li>虽然图像采集过程中添加活化缓冲液，检查亮场通道，确保油已被激活缓冲流离失所。 注意：这将首先显示为阴影，由于移液管断裂的光束路径，但也将导致在小油滴的外观。有些油滴可以与成熟的卵子，这将影响实验结果和图像质量保持联系。 </li><li>如果油没有从初始加成活化缓冲液重复的位移步骤5.4 - 50.6。 </li><li>一旦油被成功移动，使时间序列直到完成运行。 注：由于在激活卵母细胞肿胀，有的蛋商会将显着增加时激活缓冲区移出焦平面或视场。在这种情况下，停止采集并安装下一个盖玻片。 </li><li>图像序列已完成收购后，保存数据。 </li></ol> 6.收购后图像处理<ol><li>打开使用斐济（http://fiji.sc/Downloads#Fiji），或同等图像处理软件中的数据集，并选择相关文件进行分析。 </li><li>要建立采集数据的投影选择：图像&gt;栈&gt; Z-项目。 </li><li>选择开始的Z-切片和停止的Z-片，典型地整个部分。选择投影式，通常设置为“最大强度”。选中复选框“所有时间框架”来选定的设置适用于整个系列。 </li><li>如果一个以上的荧光信道已被成像，使用该频道工具来启用颜色之间的运动，使得复合材料，或者使图像的灰度。选择图像&gt;颜色&gt;频道工具。 </li><li>要调整图像亮度和对比度选择图像&gt;调整&gt;亮度/对比度。 </li><li>要导出单个图像，移动滑块时间表的有关框架，然后选择文件&gt;另存为&gt; JPEG，或任何首选格式。 </li><li>选择导出的文件的位置和名称，然后保存。 注意从成像软件的帧频将被要求提供在图像上的时间戳。 </li><li>以时间序列导出为影片选择：文件&gt;另存为&gt; AVI，然后选择帧速率（〜每秒7帧）。 </li><li>选择导出的文件的位置和名称，然后保存。 </li><li>为了节省在斐济格式调整文件，选择File&gt;保存。 </li></ol>

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The first critical step in this protocol is isolating the mature eggs without damage. This can be achieved by gentile maneuvering of the eggs with the dissecting probe. Practice will enable this manipulation to be executed without damage to the eggs. A second crucial step is avoiding the loss of the sample when activation buffer is applied to the mature egg in oil. Application of the activation buffer should be done slowly and without contact with the coverslip. This step can be challenging if the microscope set-up does not allow for an easy access to the sample. Moving axillary parts of the microscope, such as a temperature incubator, is advisable. 
Modifications to this protocol can be made in order to visualize other fluorescently tagged factors, instead of Ca2+ at egg activation. More generally, different stages of Drosophila development or the results of adding a different buffer could be analyzed using this protocol. 
Troubleshooting may be required if the settings on the microscope are not optimal for visualizing the signal from the sample. This can be achieved by increasing or decreasing the laser power, altering capture range and adjusting the Z-stack parameters. Another issue might be eggs consistently moving out of the field of view upon the addition of activation buffer. If this happens regularly, allow the mature eggs to settle on the coverslips for a longer period of time, 15 to 20 minutes. Beware that using a higher viscosity oil will alter the displacement of the oil by activation buffer. 
This technique presented here is limited by the working distance of the objective, dehydration of the mature eggs and the ability to dissect the mature eggs without damage. However, when compared to in vivo imaging methods where the mature oocyte passes through the adult female and is deposited 13, our method enables more spatial resolution, the option to physically manipulate the mature egg before activation and the ability to test the role of egg activation without fertilization. 
There are many potential future applications of this technique, including testing reporter constructs for cellular components at egg activation and visualizing the Ca2+ wave in mutant backgrounds 14,24. Together with experimental and genetic tools, the ex vivo egg activation assay presented here enables the study of the trigger, propagation and downstream effects of the universally conserved Ca2+ wave.

在蛋（卵母细胞）的激活细胞内钙的变化2+浓度是所有研究的有机体1,2的保守成分。该事件启动了广泛的Ca 2+依赖性的过程，包括细胞周期的恢复和存储mRNA的翻译。由于这一要求的钙离子 ，可视化细胞内钙离子浓度瞬时变化是关键的兴趣。 历史上，被选定为基于它们的卵的大小和可用性上激活卵子的研究模式生物。各种可视化的方法已被用于跟踪和细胞内Ca量化改变在这些系统中2+浓度，其中包括：在青鳉鱼3的光蛋白水母发光蛋白; 的 Ca 2+敏感的荧光染料如的Fura-2在海胆和仓鼠4,5;和钙绿-1-葡聚糖在爪 6。的产生和基因编码的钙指标（GECIS）改进已经改变可视化钙动力学体内 7的能力。这些遗传构建体在特定组织中表达，并限制对侵入组织标本8的必要性。 GCaMPs是绿色荧光蛋白（GFP）基类已经非常有效GECIS的，由于其高的 Ca 2+亲和力，信噪比和能力来定制9-11。在钙离子的存在下，在GCaMP复杂经历一系列的构象变化，开始与钙离子的钙调蛋白的结合，即结果在GFP元器件9的增加的荧光强度。 GCaMPs已在研究被广泛用在果蝇神经元的可视化细胞内钙12的变化。最近applicati在GCaMP技术成熟果蝇卵可视化钙离子已经揭示了蛋激活13,14一个短暂的钙波。在钙离子波可以在低倍镜下采用体外活化试验13,14 体内排卵13期间或在更高的放大倍率可视化。在体外测定中，单独的成熟卵母细胞从卵巢中分离，并用低渗溶液激活，称为活化缓冲液，这已被证明是概括在体内活化15-17的事件。 这种体外试验使不同的实验条件，包括药理中断，物理处理和基因突变下的钙波易高分辨率可视化。本文展示了成熟的卵子果蝇对体外激活和日准备用于可视化使用GCaMP的钙波ê随后的显微镜。这种方法可以用于测试的 Ca 2+波的起始和控制，并探测下游结果。

<table border="0" cellpadding="0" cellspacing="0" width="999">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dry active yeast</td>
			<td style="width:373px;">Fleischman&#8217;s</td>
			<td style="width:243px;">#2192</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>Various will work</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lamp</td>
			<td>Mircotec Fibre optic</td>
			<td>MF0-90</td>
			<td>Various will work</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Medical wipes</td>
			<td>Kimberly-Clark Corporation</td>
			<td>KLEW3020&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Marker pen</td>
			<td>Stabilo</td>
			<td>841/46</td>
			<td>OHPen universal, black - available in most stationary shops. Superfine Width 0.4mm</td>
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			<td height="21" style="height:21px;">CO2 pad</td>
			<td>&#160;Genesee Scientific</td>
			<td>59-114 (789060 Dutscher)</td>
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			<td height="21" style="height:21px;">Cover slip No. 1.5, 22 x 50mm</td>
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			<td>Menzel-Glaser-22-50mm-MNJ 400-070T&#160;</td>
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			<td height="40" rowspan="2" style="height:40px;">Halocarbon oil, series 95</td>
			<td rowspan="2">Halocarbon Products Corp.</td>
			<td>Distributor in Europe:</td>
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			<td height="20" style="height:20px;">Solvadis (GMBH)</td>
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			<td height="20" style="height:20px;">Oil 10 S</td>
			<td>VWR Chemicals</td>
			<td align="right">24627.188</td>
			<td>Can be used instead of Halocarbon oil, series 95</td>
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			<td height="20" style="height:20px;">Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>Whilst these tool should be clean, use of an autoclave is not necessary, ethanol would be sufficient.</td>
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			<td height="20" style="height:20px;">Probe</td>
			<td>Fine Science Tools</td>
			<td>10140-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass pipette</td>
			<td>Fisherbrand,</td>
			<td>FB50251</td>
			<td>Pasteur Pipettes, glass, unplugged, 150mm length&#160;</td>
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			<td style="width:373px;">Regina Industries</td>
			<td style="width:243px;">P1014</td>
			<td>GPPS&#160; 80mm x 25mm</td>
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	</tbody>
</table>

imaging calcium in drosophila at egg activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

在这里，我们已经演示了如何进行体外活化准备成熟果蝇卵。鸡蛋表达的Ca 2+动力学在蛋激活一个GECI使成像和胚胎发育的开始（ 图1）。应当注意的是，根据不同的GCaMP使用的，特别是肉豆蔻酰基的存在，结果可能有轻微的质的差异13 14。我 ​​们还证明蛋激活期间用于功能的肌动蛋白作用通过加入肌动蛋白聚合的抑制剂，细胞松弛素D，以活化缓冲液（ 图2）14。 一种在蛋激活细胞骨架的要求是守恒的。在C.线虫，受精后，细胞骨架成分改组，以制备单细胞胚胎用于第一不对称分裂21,22。在海胆，cytoskele中断以下激活TAL重新组织已表明通过防止收缩环形成23来影响细胞周期。肌动蛋白在介导的 Ca 2+波，并在蛋活化果蝇其下游功能中的作用仍有待完全阐明。 在果蝇中，可以使用此体外活化实验达到的钙波和细胞骨架的联合可视化。时间序列的多个信道的可获取和细胞内Ca 2+的动力学可以在卵母细胞（ 图3）中的肌动蛋白组织的变化相匹配。 <img alt="图1" src="/files/ftp_upload/54311/54311fig1.jpg" /> 图1：一个单一的 Ca 2+波在果蝇卵的激活时间序列的体外成熟卵果蝇表达UASt- myrGCaMP5继一活化缓冲液（AH）的ddition。在胞质钙的上升2+起源于后极（B）和传播（BF）与约1.5微米/秒的平均速度。波的启动通常是加活化缓冲液的3分钟内观察。以下激活，成熟卵子的细胞内钙水平返回到预活化水平（G，H）。见补充电影1（总时间33分钟）。比例尺= 50微米。最大投影为40μm。 <a href="https://www.jove.com/files/ftp_upload/54311/54311fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54311/54311fig2.jpg" /> 图2：加入细胞松弛素D的活化缓冲摄动的Ca2+波传播。时间序列的体外成熟卵果蝇表达UASt- myrGCaMP5下加入含10微克/毫升松弛素D终浓度（AH）的激活缓冲区。虽然钙波在后极（A）中发起的，它受到损害，并且不到达卵子（F）的前（白色箭头表示波的前部）。没有进一步的 Ca 2+变化在30分钟内观察到的）。见补充电影2（总时间30分钟）。比例尺= 50微米。最大投影为40μm。 <a href="https://www.jove.com/files/ftp_upload/54311/54311fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54311/54311fig3.jpg" /> 图3：达E肌动蛋白和钙离子的联合可视化在果蝇，时间序列体外激活GG成熟果蝇卵表达UASt- myrGCaMP5（青色）和UASP -F-Tractin.tdTomato（品红）以下除了活化缓冲液（AH）。的钙波从后极发起和恢复如在图1。肌动蛋白似乎随时间变化，白色箭头（A 对 H）。缺乏成熟卵的中心的 Ca 2+信号的检测是在图像捕获期间由于样品的移动。比例尺= 50微米。最大投影为40μm。 <a href="https://www.jove.com/files/ftp_upload/54311/54311fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Imaging Calcium in Drosophila at Egg Activation at JoVE.com

Imaging Calcium in Drosophila at Egg Activation

本文介绍了激活卵子在果蝇中可视化钙离子的适应体外协议。

在成像钙<em&gt;果蝇</em&gt;在蛋激活

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

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JoVE Journal

Developmental Biology

Imaging Calcium in Drosophila at Egg Activation

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Live-imaging of the Drosophila Pupal Eye

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Upright Imaging of Drosophila Egg Chambers

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Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

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Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

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