这项研究是由牙齿和颅面的研究国家研究所院内研究计划的美国国立卫生研究院的，支持的。

第1部分：显微镜及成像设置的制备<ol><li>任何倒置共聚焦显微镜应适合于IVM。在此研究中在Olympus IX81倒置显微镜，装备有Fluoview-1000激光扫描共聚焦单元被使用。为了达到最佳的分辨率，使用高NA的目标，如浸水UPLSAPO 60x/1.20 NA和油浸计划载脂蛋白60x/1.42 NA建议。油浸目标具有较高的收集效率，尽管它们需要使用的组织和目标之间的盖玻片，它们提供了更好的成像质量内从表面15至20微米的用于图像的区域时。另外，使用的油消除了在更长的成像会为水蒸发的关注。为了保持该温度和湿度，在显微镜应完全封闭并用温水和加湿的空气供给。可选地，动物，显微镜载物台，并且目标必须通过其他方法来加热。例如，化学热垫可以用于动物和阶段，以及专门设计的目标加热器可用于该目的（ 图1A）。客观加热器应在启动程序前的30分钟进行切换。注意：当热垫是使用以保持动物温热纱布具有放置在垫与皮肤之间。对皮肤有烧伤进行监控。 </li><li>在大多数市售的共焦显微镜的标准级具有用于插入用来容纳滑动，培养皿，并且不同尺寸的板的各种人的开口。为了进行体外成熟，标准的载物台插入35毫米培养皿上下颠倒，覆盖40毫米的玻璃幻灯片，然后在高真空硅脂或胶带（ 图1A）作抵押。自定义插件，也可以由3毫米厚的铝板制造。玻璃表面则cleaned用70％乙醇和刷用的Kimwipe。 </li><li>制作一个定制的持有人了SGS固​​定（稳定剂）。持有人的目的是为了稳定到位了SGS和严格对夫妇，他们在舞台上和盖玻片。保持器可从60毫米的塑料陪替氏培养皿进行。首先，在盘子的底部脱离的壁，切成约15×10mm的矩形。然后，边用细砂纸打磨圆润打磨。四垫片（1.5-2毫米）通过去除1毫升注射器活塞的橡胶提示建。通过使用细砂纸，隔离物的表面与四角塑料片进行研磨。的间隔物，然后通过使用凝胶状瞬间粘合剂胶接，并用细砂纸在一个平面上以后进行平滑处理。注意，对于老年动物，持有人的尺寸和间隔件可能必须增加，以适应稍大腺体。 </li><li>制备的光学耦合凝胶。 0.3％卡波姆-940（表2）凝胶是由升温100毫升的超纯水，并溶解5.47克D-山梨醇的制备。然后，0.3克卡波姆-940的加入。烧杯应覆盖并放置在搅拌下加热板（最低热定形）为几个小时，或直到所有的卡波姆团块完全溶解。最后，Trietanolamine被逐滴加入，同时轻轻搅拌，直至pH为约7.4为止。将凝胶贮存在4℃下13。 </li></ol>第2部分：动物和麻醉<ol><li>之前进行的动物实验中任何的协议，应该由当地的动物保健和伦理委员会的批准。重要的是，研究人员应受过适当训练来执行外科手术和在继续操作之前，与当地的动物保护委员会或机构的兽医应咨询。 </li><li>在这项研究中，小鼠模型（GFP / mTomato鼠）衍生的通过杂交FVB小鼠可溶性表达绿色荧光蛋白（GFP小鼠）的Wi表达的膜靶向肽融合荧光蛋白串联番茄（mTomato小鼠）第6 C57B6小鼠中使用。注意：它通常需要20到30分钟的动物成为完全麻醉，并准备向成像</li><li>小鼠被安置在一个受控制的环境中的动物设施和被允许在实验前适应环境一周。这一步是必要的成像超导重力仪，因为任何痛苦的结果增强分泌活动12。水和食物提供自由采食 。从2-5个月大的雄性用于成像实验（20-30克）。 </li><li>前麻醉，权衡动物，以确定麻醉的适当剂量。 </li><li>自诱导注射麻醉剂的施用的应力，可能会损害超导重力的分泌活性，诱导麻醉前由异氟醚吸入，接着注射麻醉剂。为了这个目标，将小鼠轻轻地进入蒸发器室中，并打开氧气流量（800-900毫米汞）。设定的异氟醚的水平至3％以启动预麻醉诱导。 </li><li>一旦动物是无意识的，注射100毫克/公斤氯胺酮的混合物20毫克/公斤甲苯噻嗪进入腹膜腔（25号针头）。麻醉剂是通过稀释100毫克/毫升的甲苯噻嗪以20毫克/毫升的盐水中，然后用氯胺酮混合而新鲜制备。该混合物进一步1:1稀释用盐水和适量是基于动物的体重注射。这种稀释有助于防止意外用药过量。注意，该混合物在20分钟后失去其有效性。 </li><li>观察动物的觉醒的迹象，如果他们（每隔一小时的初始剂量约75％在45分钟第一次注射后，50％以后）现身麻醉注入更多的麻醉剂。按捏爪子，观察反应检查麻醉深度，每隔15分钟。注意：始终管理眼药膏，以防止眼睛干涩。 </li><li>麻醉诱导体温，这会影响该实验的再现性和动物的健康。因此，鼠标应该保持下的热灯或加热垫实验的整个持续时间。注意：热灯应至少12-18英寸从动物，以防止燃烧</li><li>动物的体温必须用配备有直肠温度探头温度计监测。 </li></ol>第3部分：动物手术和定位的活体显微镜<ol><li>准备工作区所有的手术器械（ 表1），应该是干净和无菌。 </li><li>用电动推剪剃光的小鼠的颈部区域。擦拭麻醉的小鼠的颈部区域的腹侧用纱布海绵浸泡在70％乙醇中。擦干多余了的Kimwipe。 </li><li>随着沉闷的弯曲镊子举起一小块皮肤在中线（近似mately在咬肌肌肉的下端），并作一小切口（ 图1B）。 </li><li>插入剪刀到开口和通过打开剪刀片分开皮肤远离下面的组织。避免通过指向剪刀向上朝向皮肤的下侧损坏下层的腺体组织。重复此步骤几次，直到皮肤从更深的组织（ 图1B）隔开。 </li><li>用剪刀，剪出松动皮肤约3毫米宽，10毫米长的条状。皮肤应切断，使得开口所在的神经，导管及血管的压盖连接到身体的其他部分的超导重力底部附近开始。清洗伤口区域后，所得到的开口将是椭圆形的，并测量大约8×12毫米。开幕（ 图1B）后两个SGS将是可见的。 </li><li>用注射器光学耦合凝胶应用到切的中间，擦拭它往用无菌纱布外面。清洁片纱布应当用于每个擦防止引入的头发或血液进入暴露组织。直到所有的血液和松散的头发被删除重复此步骤。 </li><li>使用＃7镊子，分离结缔组织远离右侧颌下腺一路腺体的底部（或者该协议可以用在左SGS）。首先，找到SG的尖端和抢结缔组织几毫米腺体的顶端的上方。撕裂周围的腺体结缔组织不损伤其实质。 （ 图1B）。一旦腺露出，轻轻地从腺分离结缔组织。如果发生出血，用生理盐水洗血了。确保所有的结缔组织分开，腺体完全暴露。保持湿润​​的腺体，定期应用光学耦合凝胶。 </li><li>在此之前使动物的显微镜，将一块厚纱布和HEA吨包到显微镜载物台保持38±2℃的温度下化学热组的温度可以通过使内袋暴露​​于大气中的切口的大小进行调整。纱布提供从金属舞台热敷袋的一些绝缘。盖上薄薄的纱布热敷袋的顶部为好。 </li><li>将动物在其一侧（右侧为右侧腺）和上纱。与颌下腺放置在盖玻片（ 图1C）的中间位置的动物。 </li><li>动物应该固定在压盖前固定并稳定在这个位置。将一块遮蔽胶带上的鼠标的前右足的腹侧面。由丝线缝合（ 图1C，插图）挂钩鼠标的门牙。创建丝线和爪子之间的一些紧张和他们通过胶带附着走上舞台。神光应的中间自然会休息盖玻片。头部和胸部应该由塑料楔，可耦合到所述阶段被额外稳定。 </li><li>将一小片镜头清洁纸在腺体（ 图1C）。延长腺体稍放置一个定制的持有人在密封管。紧密结合的稳定剂用胶带阶段。耦合可以由一个金属夹子连接到所述阶段（ 图1C）而得到提高。紧张和尖锐的角度，应避免，因为它们可能会影响血液的流动。紧随稳定检查血流无论是在视觉上检查压盖（它应该保留粉红色着色）或通过荧光。在GFP /米 - 番茄鼠标，毛改变血液流动的可以很容易地计算，由于血管和一些血细胞的两个被标记为与m番茄。 </li><li>覆盖动物用纱布和温水垫。通过使用装有瓦特温度计测量的露出腺的温度第i小的柔性热电偶探头放置在与露出的压盖的一侧接触。 </li></ol>第4部分：成像参数<ol><li>腺体表面上使用萤光照明的重点不是通过使用GFP或RFP的过滤器设置。找到SG的表面上，并评估血流在毛细血管和组织的整体形态。组织损伤的症状包括细胞膜起泡或大液泡的外观。 </li><li>可通过落射荧光或通过预扫描选定的区域评估的组织的稳定性。一个稳定的制剂是必需的，因为大部分的运动伪影不能在后处理数据分析被删除或修正。如果准备不稳固，都需要调整。 </li><li>为了找到合适的焦平面图像预扫描模式（1X变焦）腺体，并逐渐将目标朝向盖玻片直到腺泡结构，分泌颗粒和plasma膜变得清晰可见（ 图2）。 </li><li>对于时间推移的摄像参数取决于个人的实验。对图像中的分泌颗粒的动态使用0.5-2秒/帧的扫描速度，和256×256象素用0.2微米/像素的空间尺度（视野约50微米×50微米的区域）。为了最佳地可视化的颗粒与质膜，光学厚度应为0.9-1.2微米的范围内设置。来激发GFP和上述串联番茄，人们可以使用的488纳米和561纳米的波长，分别。峰值功率被设置大约0.5毫瓦的488纳米和1毫瓦的561纳米。这将确保最小的漂白。 </li><li>探测器的设置必须被调整到的动态范围内保持的信号，并且依赖于样品的亮度。 </li><li>一旦所有的参数都设置，成像可以开始。首先，利用图被用作参考点的电场的快照。而成像时间的推移，可能会出现在XY和Z组织的漂移。这可以通过调整扫描区域和Z位置可以手动补偿。该调整应逐步由小的增量，以避免工件进行。此外，该参考图像可以被用来确定光漂白是否发生。如果显著漂白检测（荧光减少超过15％-20％），0.1毫瓦降低激光功率。 </li><li>刺激的胞外分泌，皮下注射0.1毫克/公斤异丙在盐水新鲜制备的溶液中。胞吐作用触发在注射后约1分钟。分泌颗粒与质膜融合将通过增加GFP荧光的周围的颗粒和上述串联番茄肽扩散到它们的限定膜（ 图3）被检测到。我们建议收购在同一地区3-4延时序列，每次10 min。 </li><li>成像会议结束后，安乐死的ANE通过CO 2吸入在安乐死室（10-12毫米汞柱，10分钟）sthetized动物。 </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degranulation+of+rat+salivary+glands+following+treatment+with+receptor-selective+agonists.">Degranulation of rat salivary glands following treatment with receptor-selective agonists.</a> Clin. Exp. Pharmacol. Physiol. 22, 330-336 (1995).</li></ol>

到目前为止，亚细胞结构已经拍摄主要是在体外 （ 如细胞培养）或体外 （ 即器官培养，组织切片，腺泡制剂）模型系统，往往不复述的完整的活组织6的特性。在这方面，这里提出的方法是一个重大的突破，因为它使成像的特定膜运输步骤（ 即调节的胞吐作用）的动力在活小鼠。 这个协议代表具有方面来设计用于体内成像的...

在过去的二十年活显微镜的出现和使用的荧光蛋白，导致在每一个细胞的过程可以想象的重大突破，从而推动我们的细胞生物学1的理解。这个领域已经从使用哺乳动物细胞培养是非常强大的模型系统，特别是当它涉及到实验操作中获益良多。然而，他们不经常提供复杂的多细胞生物2生物学的真实再现。这个问题已经开始通过活体显微镜（IVM）的发展来解决已经敞开了大门调查，如神经生物学，免疫学和肿瘤生物学3场关键的生物学问题。迄今为止，大多数基于IVM的研究已经进行了在组织和个体细胞的水平，而不提供关于亚细胞区室的动力学的任何信息。最近，我们的实验室和其他s有开发能力的成像亚细胞结构在活老鼠4-7，13-15，并允许在体内的药理和遗传操作的IVM技术。这种方法已被用于我们研究膜贩运体内 ，更具体内吞作用和胞吐作用调节6,7。 我们的实验模型系统是基于公开，稳定和麻醉成像啮齿动物的颌下腺唾液腺（SGS）。 SGS集团作为模型器官体外成熟的选择是由于这一事实，即腺体是通过执行一个小手术方便，可以外不影响他们生理和稳定，以减少因心跳和呼吸运动伪影。此外，SGS可以选择性基因通过在唾液腺导管8,9注射病毒性或非病毒为基础的载体操纵。最后，SGS是偏光epith组成的外分泌腺elial细​​胞，从而形成腺泡和导管，肌上皮细胞和基质细胞的复合物的人口。因为这个原因，它们是研究胞吐，胞吞作用，基因递送，和肌动蛋白细胞骨架，如在我们的最近研究10高亮显示，并提供研究细胞生物学的各个方面，如细 ​​胞极性，细胞分裂，细胞的机会的良好模型细胞连接，和离子通道。 在本文中，我们详细描述了在活老鼠的超导重力仪的上皮细胞内实现分辨率的成像协议。具体来说，我们将展示如何将图像分泌颗粒中了SGS的腺泡细胞中调节胞吐作用。如前所示，在刺激时具有β-肾上腺素能受体激动剂，分泌颗粒熔合根尖质膜和逐渐塌陷，释放出它们的内容到腺泡小管6。我们的目标是提供基本的工具来调查有m在外科手术和动物处理inimal经验，使他们能够在亚细胞分辨率成功执行IVM。由于IVM的最具挑战性的部分是动物的准备，在这里我们专注于那些用来揭露和固定了SGS不影响它们的功能基本手术过程的描述。作为标记的亚细胞结构，一些策略，比如全身递送荧光探针，使用转基因动物，或两者的组合的程序，已在别处7,11说明。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>Isoflourane (Forane)</td>
			<td>Baxter</td>
			<td>101936-40</td>
			<td>Handle under chemical hood</td>
		 </tr>
		 <tr>
			<td>Ketamine (ketaved)</td>
			<td>Fort Dodge Animal Health</td>
			<td>57457-034-10</td>
			<td>Stock solution 100 mg/ml</td>
		 </tr>
		 <tr>
			<td>Xylazine (Anased)</td>
			<td>Akorn, Decatur</td>
			<td>61311-481-10</td>
			<td>Stock solution 100 mg/ml</td>
		 </tr>
		 <tr>
			<td>Neomycin/Polymyxin B</td>
			<td>Bausch and Lomb</td>
			<td>24208-785-55</td>
			<td>Use to lubricate the eye of the mouse</td>
		 </tr>
		 <tr>
			<td>Carbomer-940</td>
			<td>Ashalnd, Inc.</td>
			<td>4607-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>D-Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Triethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>90279</td>
			<td>Add drop wise to prevent the solution to solidify</td>
		 </tr>
		 <tr>
			<td>Isoproternol</td>
			<td>Sigma-Aldrich</td>
			<td>16504</td>
			<td>Prepare fresh solutions in saline when needed. Stocks can be stored at -20 &deg;C</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Instrument</td>
		 </tr>
		 <tr>
			<td>Isoflurane V 1.9 (Vaporizer)</td>
			<td>Braintree Scientific</td>
			<td>190AF</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Portable Downdraft table equipped with HEPA filter</td>
			<td>Hazard Technology</td>
			<td>PDDT</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Heat lamp, Model HL1</td>
			<td>Braintree Scientific</td>
			<td>HL-1 US</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MicroTherma 2T Thermometer</td>
			<td>Braintree Scientific</td>
			<td>TW2</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Operating Scissors (11.5 cm straight</td>
			<td>World Precision Instruments </td>
			<td>5003708-12</td>
			<td></td>
		 </tr>
		 <tr>
			<td>#7 curved tip tweezers</td>
			<td>World Precision Instruments</td>
			<td>14187</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microscissors</td>
			<td>World Precision Instruments</td>
			<td>503365</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black Braided Silk suture #4.0</td>
			<td>George &amp; Tiemann &amp; Co</td>
			<td>160-1219-4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gauze sponges 2" x 2"</td>
			<td>Tyco Healthcare</td>
			<td>9022</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lens cleaning tissue</td>
			<td>Olympus</td>
			<td>CL-TISSUE (M97) AX6476</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

intravital microscopy for imaging subcellular structures in live mice expressing fluorescent proteins

在这里，我们描述了一个程序，在现场啮齿动物图像的亚细胞结构是基于使用共聚焦活体显微镜的。作为一个模型器官，我们使用活体小鼠的唾液腺，因为它们提供多个优点。首先，他们可以很容易地接触到使访问光学和稳定，以促进由于心跳和呼吸运动伪影的减少。这显著有利于成像和追踪小亚细胞结构。第二，大多数唾液腺的细胞群是来自器官的表面接触。这允许使用共焦显微镜，其具有更高的空间分辨率比已被用于活体成像的其它技术，如双光子显微镜。最后，唾液腺可以容易地控制药物学和基因，从而提供了一种稳健的系统在分子水平上研究生物过程。 在这个研究中，我们侧重于设计遵循在腺泡细胞分泌颗粒的胞吐作用和根尖质膜，其中分泌颗粒熔化后的β-肾上腺素能受体的刺激的动态动力学的协议。具体而言，我们所用的转基因小鼠，其共表达GFP的细胞溶质和膜靶向肽融合荧光蛋白串联番茄。然而，我们用于稳定和图像唾液腺的程序可以扩展到其他小鼠模型并耦合到其他的方法来标记在体内的细胞成分，使各个亚细胞结构，如内涵体，溶酶体，线粒体的可视化，并肌动蛋白细胞骨架。

在GFP / mTomato鼠标，腺泡显示为明显不同的结构，这表示细胞内GFP和膜定位的串联番茄肽（ 图2中 ，虚线）。在个别腺泡，腺泡细胞通过上述串联番茄肽划定。 GFP是在清晰可见的腺泡细胞内（ 图2中，箭头）的细胞核也检测到。胞质GFP被排除出现约1-1.5微米，直径为暗圆形小泡（ 图2，小图，箭头）的分泌颗粒。为了形象化胞吐的事件，我们专注于富含分泌颗?...

Watch this Scientific Journal Video about Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins at JoVE.com

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

活体显微镜是一个功能强大的工具，使活体动物成像的各种生物过程。在本文中，我们提出了用于成像的亚细胞结构，如分泌颗粒，在活体小鼠的唾液腺的动力学的详细方法。

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

intravital-microscopy-for-imaging-subcellular-structures-live-mice

Research

JoVE Journal

Biology

23.5K 次观看.  National Institutes of Health. 活体显微镜是一个功能强大的工具，使活体动物成像的各种生物过程。在本文中，我们提出了用于成像的亚细胞结构，如分泌颗粒，在活体小鼠的唾液腺的动力学的详细方法。 

视频: Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

科研

生物学

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

在活细胞荧光蛋白的双探头光学突出纯净的光转化的

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

黄荧光蛋白标记 p62 在攻击样诱导结构中光漂白后的荧光恢复

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...