我们要感谢国立卫生研究院（R15 - 03 GM065444的LB）的美国国立卫生研究院（NIH）和美国国立糖尿病，消化道和肾脏疾病研究所（KO）研究院院内研究计划的拨款。国家科学基金会奖（DBI - 0923402）出资收购LSM700共聚焦显微镜。我们还要承认一般的援助和安迪金博士的手稿上的有益的意见。幸得线虫整个社会资源共享，协议和想法。

1。线虫文化<ol><li>获取适当的C.线虫线虫遗传学联合中心（CGC）或从同事的应变。 </li><li>生长在NGM琼脂板OP50细菌草坪种子（3）线虫。分析的GFP株生长在25 ° C是建议。 </li><li>显微镜实验前的一天，到种子板块中挑选至少40 L4幼虫板放置在25 ° C过夜。这些蠕虫将实验的青壮年。 </li></ol> 2。注射如果是可取的观点，如溶酶体或线粒体的结构，成年人可以注射用荧光染料，以可视化之前。 <ol><li>准备干琼脂糖注射垫。 （这可以是一个多天前） <ol><li>准备在水熔化的2％琼脂糖。巴斯德吸管套上一个22X54毫米玻璃罩2滴。跨明智的下降顶部放置另一个玻璃罩。为了达到适当的厚度，按顶部的玻璃罩，直到垫的直径约为1.5厘米。让我们坐5-10分钟。 </li><li>卸下顶部玻璃罩。 80 ° C烘箱中放置1小时，脱水琼脂糖垫或允许坐在台式过夜。 </li></ol></li><li>准备Mitotracker或Lysotracker解决方案。 <ol><li>在鸡蛋缓冲稀释荧光剂。我们通常使用Mitotracker或Lysotracker的1:10稀释。 </li><li>注射针是由1.2外径玻璃毛细管。将注射针头，用针拉马。 </li><li>使用拉Pastuer吸管回填土用稀释后的染料的针头。 </li><li>在轻，直到用湿盒放置针。 </li></ol></li><li>预喷射：设立显微镜和针。 <ol><li>装载到注射针注射器具。我们的设备是一个Narishige直接驱动显微操纵器，安装到与DIC的成像能力配备了尼康TE200倒置显微镜阶段。 </li><li>针连接的压力调节器连接到一个1.2 mm内径管。无论是压缩空气或氮气，可用于外部压力源。设置调节到20-25 PSI。 </li><li>重矿物油（石蜡油）滴放置2到注射垫。 </li><li>注射垫放到显微镜和更低的进油针。检查从针确保流体流动自由，当施加压力时，。应用注射压力，并观察是否流体流动的针。如果没有，你需要轻轻挣脱针年底。可破针，轻轻推入注射垫放在破玻璃罩的一个小一点的提示。针后是流动的，移动到下一个步骤。 </li></ol></li><li>注射。 <ol><li>大约1个小时前观看胚胎转移到注射垫滴油年轻的成虫。执行此转让，使用解剖显微镜。使用铂丝尖为转移蠕虫与蠕虫挑。 </li><li>排列3 - 10蠕虫，使他们直接躺在垫。注射，这是最简单的，如果蠕虫一字排开彼此平行，相隔一个蠕虫病毒长度略小于。一旦蠕虫注射垫，重要的是，迅速开展工作，完成注射的过程中，蠕虫灭亡前从干燥。 </li><li>放置到注射显微镜显微镜阶段的蠕虫的玻璃罩。集中到中央部分的远端性腺管（脊柱）。然后利用显微操纵器移动到同一焦平面针尖。移动阶段的水平，使蠕虫病毒是通过针头刺破。一旦针尖脊柱内，施加压力，使性腺，以填补与染料混合物。注射注射垫所有蠕虫的两个性腺武器。 </li><li>注射后，用一把拉住巴斯德吸管，提供约0.5毫升蛋缓冲区蠕虫。这将让他们死于脱水，并让他们从注射过程中收回。 </li><li>允许蠕虫在室温下1-2小时的恢复，在光，湿盒。 </li></ol></li></ol> 3。琼脂糖观看胚胎垫<ol><li>鸡蛋中的缓冲区准备熔化的2％琼脂糖。 </li><li>用巴斯德吸管，把3滴熔化琼脂糖到一个干净的显微镜幻灯片。 </li><li>将幻灯片之间有两个标签磁带覆盖一层其他幻灯片。这些作为，这将确保您的琼脂糖凝胶垫是正确的厚度的垫片。 </li><li>垂直下降到琼脂糖滴放置一个二次清洁的显微镜幻灯片。向下按，直到顶部的幻灯片指南幻灯片的磁带在于。 </li><li>就在你准备安装观察胚胎，删除顶部的幻灯片。 </li></ol><ol><li>如果没有被注入蠕虫，请按照下面的两个步骤。否则，跳过它们。 <ol><li>板的前一天，准备选择年轻的成虫。你应该能看到里面的青壮年胚胎。 </li><li>选择到非种子选手NGM板5-20蠕虫。允许蠕虫几分钟抓取周围的非种子选手板，使蠕虫就会失去大部分被卡住他们的细菌。 </li></ol></li><li>获得成虫的幼胚<ol><li>将一个鸡蛋缓冲区5-20μL在一个18毫米的玻璃盖玻片（盖玻片厚度1.5是可取的）的中间下降。 </li><li>将产蛋缓冲区下降到蠕虫的非种子选手板或注射垫。 </li><li>削减了26号皮下注射针头的开放蠕虫。剖开外阴附近的蠕虫。您应该看到胚胎溢出的蠕虫病毒。 </li></ol></li><li>安装到显微镜玻片<ol><li>倒置显微镜幻灯片，准备用琼脂糖垫，降低到与胚胎玻璃罩。 </li><li>如果需要延长时间的推移，用凡士林密封的玻璃罩的边缘，以防止dessication。 </li></ol></li></ol> 5。显微镜<ol><li>寻找合适的胚胎。 <ol><li>将滑入显微镜阶段。 10X镜头扫描，发现胚胎。 </li><li>要发现早期阶段的胚胎，搜索完成产妇减数分裂过程中的胚胎。减数分裂的胚胎可以区分从以后的胚胎，因为他们没有经历缩短。 （胚胎完成减数分裂后，你可以看到一个很大的差距之间的细胞膜和前年底的蛋壳。） </li><li>一旦你确定了一个适当上演胚胎，将胚胎发育的记录到一个更高的放大倍率的镜头。 </li></ol></li><li>成像<ol><li>在这个例子中，我们想象胚胎中表达mCherry：H2B和GFP：泛素标记的蛋白质。我们正在使用的蔡司LSM700传统的激光扫描显微镜。 </li><li>使用63X 1.4NA透镜收集图片。 488和555激光器是用最小的激光功率和最大增益。排列堆栈是收集每10-15秒。根据漂白程度，收集30-60时间点。 ž堆栈的图像被折叠成一个最大的最后时间推移视频投影图像。 </li></ol></li></ol> 6。代表性的成果： <img src="/files/ftp_upload/2852/2852fig1.jpg" alt="图1" /> 图1施肥C.第一次有丝分裂。 线虫。 <img src="/files/ftp_upload/2852/2852fig2.jpg" alt="图2" /> 图2。显微注射过程。 补充视频1视频定时的GFP。泛加Lysotracker在减数分裂胚胎蓝<a href="http://www.jove.com/files/ftp_upload/2852/2852fig3.avi">点击这里观看视频。</a>

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没有利益冲突的声明。

与精子的融合后，受精卵经过一系列的动态活动。这些事件从一个相对静止的细胞进入迅速发育中的胚胎卵母细胞转化。在许多生物体中，细胞质重排，建立胚胎极性和卵子激活的关键。 c. 在线虫胚胎提供了一个极好的机会观察这些卵激活和胚胎发育的早期事件。胚胎是透明的和早期发育的事件时有发生较快，施肥后C。 线虫的研究人员已经产生了许多有用的表达荧光标记的蛋白在早期发展的转基因株系。与RNA干扰或突变一起使用这些菌株提供了一个功能强大的系统解剖构成一个多细胞生物体的发展（5，6，7，8）的分子途径。此视频文章介绍了一种实用的方法，使用这些工具和技术， 在 C早期胚胎发育过程中记录事件线虫。 成熟的C。线虫的卵母细胞在减数分裂前期我被捕和成人雌雄同体精囊受精。受精后，卵母细胞通过减数分裂I和II的其余部分的收益。使用荧光标记的组蛋白H2B（5），可以观察到这些阶段。在完成产妇减数分裂，精子DNA仍然简明（见图1）。完成减数分裂后，母亲和父亲的DNA变得decondensed和两个原核的形成。产妇的原核迁移对父亲的原核和他们一起加入胚胎中心附近。有丝分裂原核会议后很快随之而来。所有这些事件都发生在不到一个小时。因此，时间推移，这一系列事件的显微镜可以很容易地完成。执行这一技术需要一定线虫培养以及除了访问共聚焦显微镜的基本显微镜技能的设施。 这里介绍的例子，采用了转基因C 。 线虫应变表示两个荧光蛋白，绿色荧光蛋白：泛素和mCherry：H2B 。共聚焦激光扫描显微镜是用来观察的这两种蛋白质的动态定位。此外，我们注射荧光标记Lysotracker，可以被用来追踪溶酶体在受精后的胚胎的命运。一个标记跟踪的注入，也可以进行可视化的线粒体或局部的钙离子浓度（9）。从理论上讲，可用于注射协议，以查看任何类型的荧光标记分子在胚胎。这可能包括标记的小分子，蛋白质，脂类等在某些情况下，它可能没有必要的，以实际注入蠕虫将很容易地摄取一些，如mitotracker（10）染料标记跟踪。不过，我们已经尝试浸泡和lysotracker注射，发现注射胚胎中的可视化效果好得多。 技术方面的考虑： 此过程中提出的技术挑战，包括显微注射技术，以及在成像的难度非常早期胚胎。关于microinjections，琼脂糖注射垫的厚度是注射成功的关键。如果垫太厚，蠕虫将dessicate和迅速死亡。如果垫太薄，蠕虫将不沾垫在注射过程中。 早期胚胎，可敏感的环境条件，如温度和缓冲条件。野生型的胚胎应主动decondensation分钟左右内完成减数分裂II（4）精子DNA和原核迁移。在20分钟内，应开始第一次胞质。如果胚胎有机会接触到非常高的激光强度或过热或粉碎过程中安装，这个过程可能被打乱。这些胚胎将被逮捕的发展。如果发生这种情况，应反复实验与激光功率下降或较厚的琼脂糖凝胶垫。 线虫胚胎不分泌，直到受精后不久，他们的蛋壳。在施肥和蛋壳分泌之间的时间短，胚胎是没有生命力的母亲（11）之外。一些实验室成像在子宫内的胚胎（5，12）避免了这一挑战。成像在子宫内提供了一定的优势，如能够捕获期间或刚受精后发生的事件。然而，这项技术要求，允许深部组织成像显微镜系统的使用。如双光子或多光子系统特别适合于本（13，14）。当使用一个激光扫描或旋转盘共聚焦显微镜，最好的图像是从胚胎获得的常规，医管局已经减少成虫此处所述。 成像系统的注意事项： 共焦成像系统的类型取决于用户的需求。在一般情况下，激光扫描系统的建议，如果希望获得尽可能高的分辨率的图像。另一方面，旋转盘系统更适合于成像高度动态的过程，图像采集较快和漂白/光毒性降低。 具体的成像参数会有所不同取决于的实验设计和共焦成像系统。在多维成像，一个需要考虑的渠道，焦平面，并正在收集的时间点。由于漂白和光毒性的限制作用，可以在任何给定的实验获得的图像数量有限。因此，如果您选择，以增加收购渠道的数量，您将可能需要减少焦平面和/或时间点，反之亦然。总数将可以采集到的图像成像，以及正在使用的共聚焦显微镜的敏感性的荧光强度的影响。更激烈的荧光基团和更灵敏的仪器，将需要更短的曝光时间，从而产生更少的光漂白和光毒性。 大量或成像系统。我们将给予已在我们自己的实验室中使用的系统的详细信息。对于旋转盘共聚焦显微镜成像蠕虫胚胎，我们使用尼康TE2000U倒置显微镜配备一个60X/1.4 NA计划阿婆目标。显微镜是连接到一个横河CSU10旋转的磁盘单元，一个滨松C9100 - 13 EM CCD摄像头，并与输出光谱的应用研究LMM5激光合并模块包含四个固态激光器，在405，491，561和655 nm的。使用这个系统，我们可以捕捉一个或两个通道，6到12个焦平面，和25到50个时间点（在30至60秒的时间间隔）。一个典型的曝光时间是100至200毫秒。 用激光扫描共聚焦显微镜成像胚胎中，我们使用的Zeiss LSM700配备了2通道和4个固态激光器（405，488，555和635 nm的）。一个计划阿婆63X/1.4NA用于胚胎成像的目标，这个系统连接到一个蔡司Axio上观察倒置显微镜。该系统利用图像采集蔡司ZEN的软件还提供了有限的图像处理和分析。与两个荧光成像时，我们通常收集1-5（〜0.5微米除了焦平面），并收集了Z堆栈每15秒。集合是继续，直到发生了显着的漂白。要产生最后的时间推移视频，Z轴堆叠图像综合ZEN的软件中使用的最大的投影工具。视频AVI文件的软件出口。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>构成</th></tr><tr><td>蛋缓冲区</td><td></td><td></td><td> 5MM HEPES pH值7.2，110MM氯化钠，氯化钾4MM，5MM镁醋酸，5MM 氯化钙 </td></tr><tr><td> Mitotracker </td><td> Invitrogen公司</td><td> M - 7512 </td><td></td></tr><tr><td> Lysotracker </td><td> Invitrogen公司</td><td> L - 7525 </td><td></td></tr><tr><td>注入毛细管吸取</td><td>哈佛器械，肯特，英国</td><td> GC120F - 10 </td><td></td></tr><tr><td>针拖轮</td><td> Kopf </td><td>型号700C </td><td></td></tr><tr><td>微量器械</td><td> Tritech研究</td><td> MINJ - 1显微注射稳压器</td><td></td></tr></tbody></table>

time-lapse microscopy of early embryogenesis in caenorhabditis elegans

线虫经常被用来作为一个模型系统在早期发育过程的研究。的胚胎，遗传资源，以及转型相对容易的透明度，是素质，使 C。 线虫早期胚胎发育的很好的模型。基于激光共聚焦显微镜和荧光标记的标签，使研究人员能够遵循在胚胎发育的特定细胞的结构和蛋白质。例如，可以按照特定的细胞器，如溶酶体或线粒体，用荧光标记的染料。这些染料可以早期胚胎显微注射的方式进入成年性腺。此外，使用荧光蛋白标记，可以遵循的特定蛋白质的本地化。例子是这里展示了使用一种荧光溶酶体染料以及荧光标记的蛋白和泛素蛋白。标记的组蛋白是用于可视化的DNA，从而确定细胞周期的阶段。 GFP标记的泛揭示了泛素囊泡在早期胚胎的动态。标记的溶酶体和GFP观察：泛可用于确定是否存在泛素泡和溶酶体的共定位。一个溶酶体染料显微注射技术。用于发电transgenenic株的技术（1，2）。成像，胚胎切割出成人的雌雄同体的线虫，并安装到2％琼脂糖凝胶垫的时间推移在一个标准的激光扫描共聚焦显微镜或旋转盘共聚焦显微镜显微镜。这种方法提供的早期胚胎发育的高分辨率可视化。

Watch this Scientific Journal Video about Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans at JoVE.com

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

本文介绍的线虫胚胎发育的早期事件的可视化技术<em&gt;线虫</em&gt;。

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

time-lapse-microscopy-of-early-embryogenesis-in-caenorhabditis-elegans

Research

JoVE Journal

Biology

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

17.8K 次观看.  University of Alabama in Huntsville. 本文介绍的线虫胚胎发育的早期事件的可视化技术线虫。

视频: Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

拆装时间推移显微镜监测肌动蛋白

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells 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 ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

线虫的耗氧率测定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

延时结构照明显微镜线粒体核素的特定标签

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

使用刺激拉曼散射显微镜对 卡诺哈布迪炎电子人 中的脂质存储动力学进行无标签成像

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Methods Article

时间推移显微镜早期胚胎发育线虫