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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作者什么都没有透露。

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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medical wipes</td>
			<td>Kimberly-Clark Corporation</td>
			<td>KLEW3020&#160;</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 pad</td>
			<td>&#160;Genesee Scientific</td>
			<td>59-114 (789060 Dutscher)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slip No. 1.5, 22 x 50mm</td>
			<td>International- No1-VWR Cat=631-0137-22-50mm</td>
			<td>Menzel-Glaser-22-50mm-MNJ 400-070T&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td rowspan="2"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Solvadis (GMBH)</td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vial</td>
			<td style="width:373px;">Regina Industries</td>
			<td style="width:243px;">P1014</td>
			<td>GPPS&#160; 80mm x 25mm</td>
		</tr>
	</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. ...

imaging-calcium-in-drosophila-at-egg-activation

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JoVE Journal

Developmental Biology

Imaging Calcium in Drosophila at Egg Activation

7.8K 次观看.  University of Cambridge. 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。 

视频: Imaging Calcium in Drosophila at Egg Activation

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

直立成像<em&gt;果蝇</em&gt;蛋钱伯斯

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

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, ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

使用培养的果蝇卵室延时电影对局部细胞和组织尺度的actomyosin动力学的分析

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

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

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 processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

荧光钙成像及其后续原位杂交，用于异种性前体特征

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 ...