我们感谢Lenne的所有成员和Bertrand实验室，特别是奥利维尔相思的软件开发和热雷米Capoulade讨论。我们也感谢PICsL-IBDM成像设备，尤其是克劳德·莫雷蒂和布莱斯Detailleur技术支持。 这项工作是由ATIP补助金从国家科学研究中心（到P-FL和VB）的支持下，Labex INFORM补助（至P-FL和VB）和赠款赛诺菲 - 安万特公司（以VB）。我们承认由法新社国立德拉RECHERCHE支持法国 - 生物成像的基础设施（ANR-10-INSB-04-01，称之为“INVESTISSEMENTS德艾文莉”）。

1，设置了光板和检测路径 图1给出了光学装置的总体方案。 <ol><li>放置和固定在光学平台上的显微镜和激光。 </li><li>结合激光器488纳米，561纳米和405纳米与相应的分色镜。经过分色镜，激光器，必须精确地共同对准。注意，激光波长的光，分色镜和光学滤波器的选择是由实验中所用的特定荧光标记物的吸收光谱确定的。 </li><li>放置在声光可调谐滤波器（AOTF）。使用供应商提供的测试板来调整AOTF的频率和功率。当井对齐的，约90％的激光功率是在最大调谐如由功率计测量发射。请注意，某些激光器可以完全由软件控制，从而可以使AOTF不必要的。 </li><li>放置和固定望远镜。注意，该望远镜是由两个镜头，扩大光束到等于或大于所述照明物镜的后孔的直径的宽度。具有最小焦距的镜头应该是第一位的。 </li><li>这里，对准并固定潜望镜使向上的光路更靠近圆柱形透镜的后孔。注意，潜望镜的两个反射镜被安装的方式，其长轴是铺设在所述反射镜的倾角，以反映该完全展开的光束。 </li><li>将旁边的潜望镜退出柱面透镜。注意，该柱面透镜将光束聚焦在一个方向上并使其准直，在另一个方向上，从而形成一个光片。 </li><li>通过使用照明物镜重新聚焦所产生的光片。注意，照明目标的图像聚焦点应与上述被检测的对象的焦点重合。投射的光束，一个画面（如墙壁）在长距离上。将照明目标沿着照明光束轴，以获取有关光片的画面锐利的轮廓。 </li><li>将四线（405/488/561/638）发射滤光片在显微镜（立方体）保留的地方。 </li><li>将EMCCD相机在显微镜输出端口。 </li><li>通过在钻孔中的显微镜载物台螺丝固定在显微镜载物台的第一本手册翻译阶段。注意，平移台的边缘具有定位于约2厘米的距离显微镜载物台的中央，从而使试管居中低于检测物镜。 </li><li>固定在第一个，第二手动平移台。两个阶段的方向必须垂直（X和Y的翻译）。 </li><li>修正了在Z压电阶段前两个阶段的顶部。注意，电动载物台可以用来代替压电阶段。 </li><li>修复比色皿HOLDER（ 见图2），在各阶段的顶部。反应杯将因此被放置在照明路径的交叉点和检测路径，它们是相互垂直的。 </li></ol> 2，检查灯板<ol><li>填充的玻璃比色皿用荧光溶液，并将其放置在样品架在两个光路的交叉点。光片因此可见。它必须是水平的，并且对称/中心相对于所述检测物镜。 </li><li>取出圆柱形透镜和获取图像来测量光片的厚度。注意，光片的厚度取决于照明物镜（约3-4微米NA = 0.3）的数值孔径（NA）。注意，对于一个给定的透镜，该光板的厚度可以增加，减少有狭缝进入后孔的光束的大小。需要注意的是荧光微球可用于测量斧显微镜的IAL和横向分辨率。 </li><li>放回柱面透镜在实验开始之前。 </li></ol> 3，C的安装线虫胚胎下面的步骤被描绘于图3。 <ol><li>使用钻石标记笔切了一块10×20×1mm的载玻片。 </li><li>制备5％的细菌用琼脂在水中煮沸，并在加热块维持在60℃。 </li><li>加50微升的上述切片载玻片的一侧聚-L-赖氨酸，晾干。 </li><li>放置在板凳上载玻片和修复它。 </li><li>并列切一块滑板与固定滑动，并加入融化的琼脂顶部下降。使用另一个清洁的显微镜载玻片上，得到薄琼脂垫挤琼脂。 </li><li>让琼脂垫，以消除顶部幻灯片之前干燥。 </li><li>切在固定滑动侧面的琼脂垫，以留下过量的大约3毫米的Of琼脂和轻轻地取出固定滑动。 </li><li>加入50微升的琼脂聚-L-赖氨酸，并让它完全干燥。 </li><li>把妊娠蠕虫在M9培养基在手表玻璃。 </li><li>切断蠕虫在用手术刀外阴的水平，以释放胚胎。 </li><li>下一个双目确定胚胎在感兴趣的阶段，通过使用微细吸管收集它们。 </li><li>将胚胎在涂有聚-L-赖氨酸是突出于切一块载玻片的琼脂的一部分。将胚胎仅次于幻灯片的边框。在琼脂的边界不定位胚胎，因为他们将没有足够的稳定。轻轻地取出的液体，以使胚胎会粘在聚-L-赖氨酸层上。 </li><li>快速对齐胚胎，以避免干燥。 </li><li>放置在塑料培养皿切割滑动，并轻轻加的M9培养基中以覆盖滑动。 </li><li>检查下的蛋都还停留在琼脂双目。 </LI&gt; </ol> C. 4。记录线虫发展<ol><li>修正了先前的准备（切幻灯片，琼脂加胚胎）在样品架上，并把它放在一个杯中。 图4描述了自制的支架和图5显示了在观察范围内的比色皿的景色。 </li><li>固定在压电阶段的反应杯。 </li><li>确定与明视场照明胚胎的位置。 </li><li>启动国产软件控制AOTF中，相机和压电阶段。请注意，一个免费的软件，如事无巨细，也可以使用。 </li><li>选择激光线和电源（AOTF）。 </li><li>使用3个方向舞台支撑圆柱透镜和照明透镜调节光片的位置。移动载物台沿横向方向（X），以获得最亮的信号。调节光片的再Z位置和纵向位置（Y），以获得第Ë最好的信噪比。注意，光片焦点的位置可能需要进行调整，从一个胚胎到另一个，以纠正光路长度的差异。 </li><li>获取的时间推移的图像，选择曝光时间，增益，两个图像，以及将所获取的图像的数量，以及激光功率之间的时间。 2颜色获取，选择第二个激光线。两个彩色图像是连续获得的。 </li><li>为了获得AZ堆栈，也可以选择和开始和结束的压电岗位的两片之间的距离。 </li></ol>

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作者什么都没有透露。

本协议描述了一个简单的安装光片镜检为C的体内成像线虫胚胎。需要创建并对准光片，包括激光器，光束扩展器，准直和聚焦透镜的光学元件，可以很容易地安装在光具座上。在与一个堂堂正正的显微镜检测路径和摄像机组合，这提供了一个简单的解决方案来设置一个光片显微镜。 这种几何形状使得样品环境简单。的活体被放置在玻璃比色皿，其被保持，定位并通过一小部分的机械件移动。作为钢板照明显微镜是切片能力的宽视场技术，只有电动单轴运动所需的3D成像。这限制了同步的要求，以一个单一的扫描元件，便于软件的开发。相较于iSPIM 6我们直立GEOMETRY允许使用高NA物镜的进行检测。相比SPIM的设置，不同的物镜在水平面内样品的安装和一系列胚胎的成像更容易。总之，这一技术的实现是简单的并且可以与小的专门知识光学来实现。这是一个更便宜的替代的共焦显微镜具有更高的速度和降低光毒性，但较低的轴向分辨率的缺点的优点。 我们还开发了一个简单的和可重复的方法来安装C。线虫的胚胎在体内使用这个系统的成像。在安装过程是快速的（15分钟），并且是适合于日常的实验。对于C。线虫成像，这种简单的设置的优点是至少两个褶皱：（i） 于全盘成像是可能的，不旋转，因此三维重建是直接的; （ⅱ）样品的安装是容易的，多个胚胎可以顺序成像在同一张幻灯片。我们发现时间推移电影的例子有足够的时间分辨率来观察细胞分裂和细胞形态的变化。光薄片显微镜来研究下的权力线虫发展也已最近由其他人6，7，8所示，因此，这种技术将是非常有用的，研究C的形态发生和图案形成线虫胚胎。 可这个系统也可以用来分析其他模式生物的发展？认识到这一点很重要薄片照明显微镜的小型和透明生物体（如C）的实施线虫是比较大的生物体如果蝇胚胎不那么严格， 在果蝇的托托成像通常需要旋转和多视图重建9,10。激励从两侧用共线激发物镜也可以降低所造成的attenuat的阴影效果离子灯11。然而，这里介绍的设置仍然适用于果蝇胚胎的图像一侧，具有低白化，低光毒性。这种设置也可能是有用的图像和小的胚胎透明，如海鞘类动物或斑马鱼。

一个生物体的形成和塑造涉及细胞运动，细胞形态变化，细胞分裂和细胞分化。了解这些过程的进展和需要协调三个维度的能力和亚细胞分辨率快速成像工具。其中包括与这些体内成像能力的技巧，光板照明显微镜也被称为单/选择性平面照明显微镜具有生产低光毒性作用，低光漂白1,2的独特优势。 在片材的照明显微镜，将样品从侧面由一个光片，它执行光学切片照亮。照明路径和检测路径是彼此垂直和光片对齐到与检测物镜的物方焦平面重合。样品被放置在两条路径相交，一个ND可沿检测轴移动，让3D成像。感兴趣的唯一的平面被照亮，并在同一时间被检测到该平面的所有点。这显著增加了采集速度，并降低光致漂白和光毒性相比，共聚焦显微镜3。 实际上，板材照明显微镜是易于安装和对齐：对于二维成像，无​​扫描是必要的既不是为了检测部分，也没有照明部;作为与切片能力宽视场技术中，三维成像可以由平台保持的样品的单一轴的移动来获得。 在这里，我们在C的全盘成像提出一个简单的光薄片显微镜及其实施的设立为线虫胚胎。C.线虫的胚胎非常适合生活在体内 IMA更改，由于其透明度，不变的血统和千篇一律的细胞定位4,5。所开发的光片的设置是基于一个标准的直立显微镜的检测。下面介绍的协议详细介绍了如何构建和调整激励模块/路径，测试及表征光片和安装样品三维成像。它还提供了对表达荧光标记不同菌株的设置收购时间推移电影的例子。

<table border="0" cellpadding="0" cellspacing="0" width="812">
	<tbody>
		<tr height="22">
			<td>SPIM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Detection</td>
		</tr>
		<tr height="22">
			<td>upright microscope</td>
			<td>ZEISS</td>
			<td>Axiophot 1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>100xW objective (NA 1.1, W.D. 2.5mm)&#160;</td>
			<td>NIKON</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>EMCCD back illuminated</td>
			<td>ANDOR</td>
			<td>DU-885K-CS0-#VP</td>
			<td></td>
		</tr>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Illumination</td>
		</tr>
		<tr height="22">
			<td>LASER 488nm / 60mW</td>
			<td>TOPTICA</td>
			<td>iBeam smart</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>LASER 405nm / 120mW</td>
			<td>TOPTICA</td>
			<td>iBeam smart</td>
			<td></td>
		</tr>
		<tr height="34">
			<td>LASER 561nm / 500mW</td>
			<td>COBOLT</td>
			<td>JIVE 500</td>
			<td>&#160;for time lapse imaging, 50 or 100mW is enough</td>
		</tr>
		<tr height="22">
			<td>filter Quad line laser rejectionband ZET 405/488/561/638</td>
			<td>AHF</td>
			<td>F57-405</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Dichroic 405</td>
			<td>AHF</td>
			<td>F38-405</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Dichroic 488</td>
			<td>SEMROCK</td>
			<td>Di01-R488</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>AOTF</td>
			<td>AA</td>
			<td>AOTFnC-400.650-TN</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>mirror</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>lens 50mm</td>
			<td>THORLABS</td>
			<td></td>
			<td>telescope 5X</td>
		</tr>
		<tr height="22">
			<td>lens 200mm</td>
			<td>THORLABS</td>
			<td></td>
			<td>telescope 5X</td>
		</tr>
		<tr height="22">
			<td>periscope</td>
			<td>THORLABS</td>
			<td>RS99/M</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>cylindrical lens f=100mm</td>
			<td>THORLABS</td>
			<td>ACY254-100-A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>10X NA 0,3</td>
			<td>NIKON</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>xyz stage</td>
			<td>NEWPORT</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Sample</td>
		</tr>
		<tr height="22">
			<td>Bacto Agar</td>
			<td>Bectkton Dickinson</td>
			<td>214010</td>
			<td>5% in water</td>
		</tr>
		<tr height="22">
			<td>Poly-L-Lysine solution&#160;</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Capillaries GC100F-10, 1.0mm oD, 0,58mm ID, 100 mm L</td>
			<td>Havard Appartus</td>
			<td>30-0019</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Aspiration tube (mouth pipette)</td>
			<td>Dutsher</td>
			<td>55005</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>M9 medium:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>KH2PO4 (3g/L)</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Na2HPO4 (6g/L)</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>NaCL (5g/L)</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>MgSO4 (1mM final)</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>cuvette holder</td>
			<td>HOME MADE</td>
			<td></td>
			<td>made by Brice Detailleur&#160;</td>
		</tr>
		<tr height="22">
			<td>sample holder</td>
			<td>HOME MADE</td>
			<td></td>
			<td>made by Claude Moretti&#160;</td>
		</tr>
		<tr height="22">
			<td>x stage</td>
			<td>NEWPORT</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>coverslip 50x20x1mm</td>
			<td>MARIENFELD</td>
			<td></td>
			<td>cut then in 10x20x1mm</td>
		</tr>
		<tr height="22">
			<td>piezo electric stage with amplifier/controller</td>
			<td>PI</td>
			<td>P-622.ZCD / E-625.CR</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>cuvette 40mmx40mmx10mm</td>
			<td>HELLMA</td>
			<td>704.002-OG.40 mm- 10mm high</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Diamond Point Glass Marking Pencil</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr height="80">
			<td>C++ (Qt) home made software</td>
			<td></td>
			<td></td>
			<td>developed by Olivier Blanc and Claire Chard&#232;s, available on request. Alternative solutions: micromanager or labview&#160;</td>
		</tr>
	</tbody>
</table>

setting up a simple light sheet microscope for in toto imaging of c. elegans development

快和低光毒性成像技术是先决条件，研究生物的全盘发展。轻质板材基于显微降低光漂白和比较，激光共聚焦显微镜光毒性作用，同时提供3D图像与亚细胞分辨率。这里我们提出了一种基于光片的显微镜，它是由一个直立显微镜和一小部分的光片的产生光电机械元件的安装。该协议描述了如何建立，对准显微镜表征光片。此外，它详细介绍了如何实现该方法在C的全盘成像线虫使用一个简单的观察室的胚胎。该方法允许的3D两色时间推移电影在几个小时内发展的捕获。这应该减轻细胞形态，细胞分裂和蛋白质标记在很长一段时间的跟踪。

从三个不同C.三3D延时录制的实验线虫菌株示出的数据，可以使用上述的片光显微镜的设置获得的类型。我们检查了这些条件下，胚胎发育正常速度和生存的成像，与以前的报告表明SPIM成像造成三只光毒性低的水平相一致线虫胚胎6,7。 第一株表达融合到绿色荧光蛋白（zuIs178; stIs10024）的组蛋白。在2小时内进行与一叠20片（切片1微米之间的距离）取每37秒记录， 图6示出在不同时间点在堆栈中的一个平面上。这两个interphasic核（箭头）和浓缩的染色质丝分裂（箭头）都清晰可见。 第二应变表达融合到绿色荧光蛋白（ruIs57）微管蛋白和组蛋白融合到的mCherry（itIs37; stIs10226）。用一叠20片（切片1微米之间的距离）取每105秒时16分钟，进行记录， 图7示出了堆叠和不同的时间点的不同平面上。相关电影1显示三维重建在连续3个时间点。在除法中，有丝分裂纺锤体（绿色）和稠合的染色体（红色）是清楚可见的，允许细胞分裂取向的发展过程中的跟踪。 第三个菌株表达载脂蛋白VIT-2融合到绿色荧光蛋白（pwIs23）和绑定的mCherry（ltIs44）的膜。用一叠10片（切片1微米之间的距离）取每30秒在25分钟内进行记录， 图8示出了叠层和不同的时间点的不同平面上。卵黄脂蛋白颗粒（绿色）的细胞内的快速运动可以很容易地其次。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig1highres.jpg" width="500" /> 图1：SPIM光学装置的照明和检测单元，前视图和底视图组成 。在照明单元中，激光通过分色镜组合输入AOTF中，它控制每个激光器的功率独立。然后，该望远镜由4倍增加的光束的大小和潜望镜把它到显微镜的高度。柱面透镜构成的光片，它是由所述照明物镜重新聚焦。检测单元集成在直立显微镜和主要由检测透镜，过滤器，管透镜和EMCCD。样品被放置在照明和探测路径之间的交叉点。压电阶段允许样品垂直（Z轴）的位移三维采集。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig2highres.jpg" width="500" /> 图2：试管支架夹持器由3个铝板和拧在该压电阶段。玻璃比色杯是由一个弹簧（红色）持有。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig3highres.jpg" width="500" /> 图3：C的安装协议线虫胚胎SPIM实验 。甲切一块玻璃上涂有聚-L-赖氨酸。琼脂的垫被定位在涂层表面。聚-L-赖氨酸，然后加入到琼脂板。最后，C。线虫胚胎在聚-L-赖氨酸包被的琼脂垫对齐。 图4：样品架 。保持器装配到玻璃比色皿的内部尺寸。两个弹簧（红色）保持载玻片（ 图3中描述）。这架然后把玻璃比色皿内。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig5highres.jpg" width="500" /> 图5：。样品架在观察范围内胚是根据协议概括在图3中固定于载玻片上的滑动是通过图4中所描述的样品架保持的样品架被插入到玻璃比色皿，由图2中所描述的保持器保持。光片是水平的，我lluminates从侧面胚胎。检测物镜是垂直的，该样品的上方。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig6highres.jpg" width="500" /> 图6：记录一个C的线虫胚胎表达的组蛋白:: GFP融合转基因胚胎的单一平面影像表达一种组蛋白:: GFP融合（zuIs178; stIs10024）在不同时间点的影像从3D延时录制提取：。栈由20片（切片1微米之间的距离）;每片曝光时间：30毫秒;堆栈之间的时间间隔：37秒;总采集时间：2小时。箭头：interphasic核，箭头：浓缩的有丝分裂染色质。在t = 0分钟的胚胎在2 - 细胞阶段和在t = 120分钟的胚胎含有约70细胞在正常发育S作为预期尿尿（注意，只包含在一个单一平面内的细胞是可见的图像，而不是胚胎的全部细胞）。照明的功率为30μW在488纳米（在照明目标的出口测量）。 <a href="https://www.jove.com/files/ftp_upload/51342/51342_LenneBertrand_Figure6.avi" target="_blank">请点击此处观看该视频。</a> <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig7highres.jpg" width="500" /> 图7：记录一个C的线虫胚胎表达微管蛋白:: GFP融合和组蛋白:: mCherry的融合。从C的3D延时录制图像中提取线虫胚胎表达微管蛋白:: GFP融合（ruIs57）和组蛋白:: mCherry的融合（itIs37; stIs10226）。拍摄条件：收购一摞20片（1片之间的距离微米），每105秒;总采集时间：16分钟;曝光时间：200毫秒为每个通道。 面板显示10片相同的堆栈。 图 b显示了相同的片每210秒。照明的功率为30μW和300μW，为488和561 nm的激光器，分别为（在照明目标的出口测量）。 <a href="https://www.jove.com/files/ftp_upload/51342/51342_LenneBertrand_Figure7.avi" target="_blank">请点击此处观看该视频。</a> <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig8highres.jpg" width="500" /> 图8：记录一个C的线虫胚胎表达VIT-2 :: GFP融合和膜针对性的mCherry。图片从C的3D延时录制提取线虫胚胎表达VIT-2 :: GFP融合（PWIS23）和一个膜定位的mCherry（ltIs44）。拍摄条件：收购一摞10片（1片微米之间的距离），每30秒;总采集时间：25分钟;每个通道曝光时间：200毫秒。 面板显示了相同的堆栈的5第一片。 图 b显示了相同的切片每30秒。 <a href="https://www.jove.com/files/ftp_upload/51342/51342_LenneBertrand_Figure8.avi" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development at JoVE.com

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

本协议描述了一个光片显微镜的建立和实施对C的体内成像线虫胚胎。

设置简单的轻表为显微镜<em&gt;在托托</em的&gt;成像<em&gt; C。线虫</em&gt;发展

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

setting-up-simple-light-sheet-microscope-for-toto-imaging-c-elegans

Research

JoVE Journal

Biology

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

23.1K 次观看.  UMR7288 CNRS, Aix-Marseille Université. 本协议描述了一个光片显微镜的建立和实施对C的体内成像线虫胚胎。 

视频: Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

光镜下的主要组成部分

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

测量在C.elegan的RAN肽毒性

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures&#160;similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet&#160;fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

样品室光片荧光显微镜中多个昆虫胚胎的同步实时成像

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.