支持部分由R01NS064593和R21ES016636（KV）。显微镜进行VCU - 神经生物学与解剖学显微镜设备，部分支持，资金从NIH-NINDS中心的核心授予5P30NS047463的。旋转盘共聚焦显微镜购买的与NIH-NCRR的奖（1S10RR027957）。

A.细胞制备<ol><li>正常人体主要的成纤维细胞（GM02270）卡瑞尔细胞储存库，卡姆登，新泽西州，并与hTERT 6永生。细胞生长和扩大在蜂星6-cm培养皿在媒体（4毫升）组成的MEM培养基与20％的胎牛血清（GIBCO），的non-essential/essential氨基酸，维生素，丙酮酸钠，和青霉素/链霉素（HyClone公司）。 </li><li>人胚胎肾293（HEK293）细胞，从美国典型培养物保藏中心获得的，并在蜂星6-cm培养皿中生长和扩大。中，细胞保持在介质（4毫升）中组成的DMEM培养液与10％牛胎儿血清，非必需氨基酸，L-谷氨酰胺和青霉素/链霉素。 </li><li> 53BP1 mCherry（N-MYC-53BP1 WT pLPC的普罗Addgene质粒19836）（pCLNR H2BG; Addgene质粒17735）和H2B-GFP融合基因的结构也表达嘌呤霉素或G418抗性基因，分别转（fibrobla针）或转染（HEK293）使用SuperFect（Qiagene）进入细胞，并保持在药物选择。荧光定期检查维护。 </li><li>图像采集前24-48小时，将细胞用胰蛋白酶消化成单细胞悬液，并以较低的密度接种到的3.5厘米FluroDish玻璃底板。 </li></ol> B.显微镜设置和图像采集该协议被开发使用的蔡司细胞观察SD旋转盘共聚焦显微镜配备了一个AxioObserver Z1架，双通道Yokagawa CSU-X1A 5000旋转的磁盘单元，配光QuantEM 512SC EMCCD相机，HXP 120C光纤为基础的照明灯，4激光器（一Lasos 100mW的多行，氩[458，488，514纳米，50 mW的405 nm二极管，40 mW的561 nm二极管，和30 mW的635 nm二极管），AOTF，一个Pecon的的XL multiS1阶段孵化体系，蔡司孵化模块（模块S O 2，CO 2 TempModule模块S，S，供热机组XL小号）和前个月XY torized阶段与一个NanoScanZ的压电Z插入。为了尽量减少的活细胞成像的球面像差，而在水性介质中，一个C-复消色差63x/1.20水/更正物镜和蔡司Immersol W浸没流体（具有折射率n = 1.334）的支持。对于2通道共焦成像中，RQFT 405/488/568/647分色镜和BP525/50（绿色）和BP629/62（红色）排放过滤器被使用。所用的系统软件与AV4 Multi-channel/Z/T，快速成像，生理学，MosaiX，标记和查找，双摄像头，里面4D，自动对焦和3-D反褶积模块：蔡司Axiovision（版本4.8.2.0）。 <ol><li>确保运行，CO 2气体的CO 2孵化系统的模块。如果在显微镜防振空气表支持的，打开空气供给（或N 2气体）的空气表。 </li><li>打开显微镜支架的电源开关，旋转式磁盘装置，数码相机，孵化模块，HXP照明灯，电动载物台，氩气激光，和计算机。 </li><li>经过1分钟的热身时间，将点火钥匙的氩离子激光器为“开”。 </li><li>在蔡司激光控制面板中，打开要使用的激光线的开关。 </li><li>拨动开关切换为“待机”，“激光运行”，的Lasos氩离子激光器控制器，调光控制器的最佳水平（ 即正下方的绿色指示灯会变成红色）。 </li><li>启动AxioVision软件。 注 ：是专门针对特定的显微镜和组件，它控制的窗口和下拉菜单，可自定义的用户界面AxioVision。因此，每个系统都有可能是唯一的接口。因此，在随后的步骤中提供软件操作的一般说明，而不是特定的软件窗口，标签和/或下拉菜单的方向。 </li><li>成像前约1小时，在软件中，定位控制的孵化器和上部腔室的加热和镜台板的开启。把温度设置为37℃。打开的CO 2的控制和设置在5％的水平。 </li><li>选择用于成像的物镜。在这项研究中，63x/1.20 NA C-复消色差水/更正物镜使用注 ：对于这样的镜头，需要浸没介质的折射率类似的水（蔡司Immersol W浸没流体）。 </li><li>如果在一个长的时间内将被采样的多个单独的位置，某些应用足够量的液浸介质中，以确保它从一个位置到另一个进行。 </li><li>放置在舞台上的菜带来物镜成与底部接触。 </li><li>在显微镜控制或软件，直接发出的光目镜，选择“适当的广角过滤器的设置荧光信号的利息（这项研究中，”红“过滤器设置53BP1和”绿色荧光蛋白“过滤器设置为H2B ）。/ P&gt; <li>查看通过眼透镜，图像聚焦，细胞中找到一个合适的领域。 </li><li>使用软件或显微镜控制，直接发射的光远离共焦旋转圆盘​​单元到端口的目镜。 </li><li>在软件中，打开相应的激光（这项研究中，561纳米激光53BP1和H2B的氩离子激光器的488 nm线）。 </li><li>对于每个通道，调整的激光的强度的声光可调谐滤波器（AOTF）控制通过调节到适当的水平。 </li><li>选择适当的的分色镜（RQFT 405/488/568/647）和发射滤波器（BP62分之62953BP1和BP五十○分之五百二十五的的H2B）。打开快门纺丝盘单元。 </li><li>选择“实时”窗口，显示当前的视野。 </li><li>在软件中，打开“相机”控制，选择相机使用（如果是双相机系统），并设定曝光时间约为100毫秒。调整％和EM获得其他埃森。 注：53BP1 mCherry结构显得有些暗淡。我们发现它有利于提高EM增益。 </li><li>在软件中，打开控制的共聚焦转盘单位和调整转盘的速度进入相机的曝光时间，捕捉到一个合适的图像（ 例如 〜100毫秒）。单击“设置”锁定的变化。 </li><li>打开“多维收购”。选择通道“选项卡，加载/选择合适的渠道。在这项研究中，我们将使用红色荧光蛋白（561 nm激光激发和BP 629/62过滤器的排放）和GFP（488 nm激光激发和BP525/50过滤器的发射线）定义的通道。 </li><li>为了确保图像配准中，一个共同的分色镜（RQFT 405/488/568/647）用于两条通道。设置软件的“自动对焦”。 注意 ：工具→设置编辑器调整的MDA设置。确保有足够的激光功率设置（“足够的激光功率”是指设置，让您激发适当的荧光基团，同时最大限度地减少光漂白）。 </li><li>在“MDA”窗口中，选择“Z-Stack的标签。选择“Z-堆栈在当前的焦点位置”。设置范围〜10微米的Z-Stack，然后选择“最佳”的一些步骤，以确保奈奎斯特采样到Z 注意：确切的Z-Stack的大小将取决于你的细胞的高度。相应的调整。 </li><li> 23。单击“开始”，产生的Z-Stack的图像进行分析，以确保设置适合你调查（ 例如在这项研究中的，这是非常重要的，整个核）。 </li><li>选择“T”（时间）选项卡。对于喜树碱我们的实验中，我们设置了成像时间点之间的时间间隔为5分钟和1小时的控制（非处理的细胞）视频会议期间的整体。 注意：尽量减少光漂白细胞，实验应该被配置为使得在AOTF激光成像的时间点之间的空白。 </li><li> F多点成像几个细胞的菜，在MDA“窗口中，选择”位置“选项卡。确保“应用”设定时间点的每个位置前/后“被选中。 ，选择“Mark_Find”。使用“现场”视图，将周围的菜，并选择合适的视野。 </li><li>多维收购菜单中单击“开始”，开始进行实验，并记录控制视频（未经处理的细胞）。 </li><li>控制视频后，添加适当的处理（在这种情况下，10μM喜树碱）。我们记录了实验（药物处理的细胞）的视频在5-10分钟的时间间隔为2-4小时。 注：要考虑的是速度，一个给定的效果，治疗后出现的一个重要问题。在视频中可以看出，53BP1灶开始〜5分钟后，加入CPT。虽然一个相对容易的过程，手动添加药物的菜和重新校准的显微镜，确实需要时间。设计实验时，必须考虑到这。 </li><li>为了监测米itosis，我们的间隔设为7.5分钟，并记录为4-5小时（特定的设置取决于使用的细胞系和其细胞周期的长度）。可能需要进行调整，在较长的录音，以防止光漂白。 </li></ol> C.图像处理与分析 Volocity软件（珀金埃尔默），该协议的开发利用。获得这些数据（AxioVision）所使用的软件也有能力来处理和分析图像。我们鼓励用户利用该软件提供给他们，并查阅相关文献，对他们的应用程序。 <ol><li>打开Volocity软件。创建并命名一个新的库，并导入你的视频文件。 </li><li>要查看的文件，我们通常会发现使用“扩展聚焦方式”设定有极大的帮助。这叠加的Z-Stack片，此协议，使我们能够可视化53BP1灶在不同领域的核。 </li><li>在必要时调整的影片。 VOlocity配备了一系列的工具，以提高采集到的图像质量。很多时间通过确保在显微镜上的设置是合适的，可以被保存在编辑以后。通常情况下，它是有帮助，反卷积您的图像，调整亮度/对比度。实验的基础上，你的特定的编辑需求会有所不同。 </li><li>添加一个相对时间戳和比例尺。 </li><li>要查看单元格中的3-D，切换到“3-D不透明度”设置。这允许在空间的3-D渲染细胞的旋转，从而提供利益的结构的细胞内的多个角度。 注：这是有助益的可视化细胞在不同的平面中，以确定结构感兴趣的旅行。例如，在“扩展聚焦方式”设定，它是很难辨别53BP1不，其实，游离在有丝分裂过程中从DNA。然而，这是显而易见的，在3-D。 </li><li>动画和静止图像可以导出成多种文件类型，根据用户的喜好。 </l<html> I&gt; </ol> D.代表性的成果响应对CPT 53BP1灶形成的一个例子的图1中所示。中华映管灶的细胞暴露在5-10分钟范围内，并保持这些病灶在整个持续时间的记录。正如在图2中所示，53BP1解离染色质在有丝分裂的发病，冷凝染色体周围形成薄膜的雾度。末期发生有丝分裂结束，53BP1再次聚集成不同的病灶。虽然HEK293细胞没有接触到CPT，但它们形成丰富的，自发的53BP1所产生的内源性DNA损伤的修复病灶。这一观察结果使我们得出结论，53BP1没有形成病灶早期有丝分裂过程中，在与以前的报告显示，类似的效果后，细胞暴露于电离辐射和radiomimetic的药物5。 从图1“SRC =”/ files/ftp_upload/4251/4251fig1.jpg“/&gt; 图1。喜树碱（10μM）引起DNA双链断裂和向培养基中添加药物后的30分钟内，在骑自行车的成纤维细胞的53BP1灶形成。 <img alt="图2" src="/files/ftp_upload/4251/4251fig2.jpg" /> 图2。，53BP1并不构成灶在HEK293细胞中有丝分裂过程中，直到telophase/G1。

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没有利益冲突的声明。

维持基因组的完整性，细胞的生存是至关重要的。如果保存的基因组会导致过早衰老，癌变，死亡8。有浓厚的兴趣，挑剔的DDR功能，源于其基础和临床研究的重要性。多年来，以帮助在细胞如何检测和修复DNA损伤的研究已开发了许多技术。传统的方法，如免疫细胞化学和西方墨点法领域的支柱，尽管最近的技术进步已经使日益复杂的方法发展。活细胞成像，在该协议中详细说明，使我们?...

<table border="1"><tbody><tr><td> 产品 </td><td> 公司 </td></tr><tr><td>陕西蜂星的培养皿</td><td>格雷纳生物一</td></tr><tr><td> FluroDish玻璃底菜</td><td>世界精密仪器公司</td></tr><tr><td> MEM媒体</td><td> GIBCO </td></tr><tr><td>非必需氨基酸</td><td> GIBCO </td></tr><tr><td>氨基酸</td><td> GIBCO </td></tr><tr><td>维生素</td><td> GIBCO </td></tr><tr><td>丙酮酸钠</td><td> Invitrogen公司</td></tr><tr><td>青霉素/链霉素</td><td>胎牛血清</td></tr><tr><td>胎牛血清</td><td> GIBCO </td></tr><tr><td> N-MYC-53BP1 WT pLPC的普罗; 质粒19836 </td><td> Addgene </td></tr><tr><td> pCLNR H2BG质粒17735 </td><td> Addgene </td></tr><tr><td> SuperFect </td><td> Qiagen公司</td></tr><tr><td>蔡司观察SD细胞成像系统</td><td>蔡司</td></tr><tr><td> AxioVision（版本4.8.2） </td><td>蔡司</td></tr><tr><td>蔡司Immersol W油</td><td>蔡司</td></tr><tr><td> Volocity软件（版本6.0） </td><td>珀金埃尔默</td></tr></tbody></table>

two- and three-dimensional live cell imaging of dna damage response proteins

双链断裂（DSBs）是最有害的DNA病变的细胞可能会遇到。如果未修复，DNA双链断裂港口的巨大潜力产生突变和染色体畸变1。为了防止这种创伤催化基因组的不稳定性，这是至关重要的，检测DNA双链断裂的细胞，激活DNA损伤反应（DDR），并修复DNA。解除武装，复员和重返社会工作的刺激时，通过触发细胞周期阻滞，以便维修发生或强制的细胞发生凋亡，以维持基因组的完整性。 DSB修复发生的主要机制通过非同源末端连接（NHEJ）和同源重组修复（HRR）（评论2例 ）。有许多的蛋白质，其活动必须精确精心策划的解除武装，复员和重返社会正常运行。这里，我们描述了一种方法，2 - 和3 - 维（D）的可视化这些蛋白质之一，53BP1。 p53结合蛋白1（53BP1）定位于地区DNA双链断裂的修饰组蛋白3,4结合，形成病灶在5-15分钟内5。组蛋白修饰和招聘53BP1和其他DDR蛋白DSB网站被认为是染色质的结构重排，以方便周边地区的损失，并有助于DNA修复6。除了 ​​直接参与修复，额外的角色被描述为53BP1在解除武装，复员和重返社会，如调节内-S检验点，G2 / M检验点，并激活下游DDR蛋白7-9。最近，人们发现，53BP1没有形成病灶在有丝分裂过程中DNA损伤，而不是等待细胞进入G1前定位附近的DNA双链断裂6。如53BP1 DDR蛋白质已被发现与有丝分裂的结构（如着丝粒）相关联的进展通过有丝分裂10期间。 在这个协议中，我们描述了使用2 - 和3-D的活细胞成像的可视化53BP1灶形成的DNA损伤剂喜树碱（CPT），以及53BP1在有丝分裂过程中的行为。喜树碱是拓扑异构酶I抑制剂，主要引起DNA双链断裂DNA复制过程中。要做到这一点，我们使用先前描述的53BP1 mCherry荧光融合蛋白结构的53BP1的蛋白质结构域的能够结合DNA双链断裂11。此外，我们使用组蛋白H2B-GFP的荧光融合蛋白的构建，能够监控整个细胞周期，但在特定的染色质的动态，在有丝分裂12。活细胞成像技术在多个方面，是一个很好的工具，以加深我们对DDR在真核细胞中的蛋白质的功能。

Watch this Scientific Journal Video about Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins at JoVE.com

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

该协议描述了一个可视化的DNA双链断裂以及其定位在有丝分裂过程中DNA损伤激活的信号蛋白的方法。

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

two-three-dimensional-live-cell-imaging-dna-damage-response

Research

JoVE Journal

Biology

14.0K 次观看.  Virginia Commonwealth University. 该协议描述了一个可视化的DNA双链断裂以及其定位在有丝分裂过程中DNA损伤激活的信号蛋白的方法。

视频: Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

科研

生物学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

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 ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

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 ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

老鼠胎儿整肠文化系统<em&gt;前体内</em&gt;信号通路和绒毛开发的三维实时成像的操作

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

三维类器官和球状体模型的染色和高分辨率成像

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.

This protocol presents a new reporter gene system and the experimental setup to detect transcription at DNA double-strand breaks with single-molecule sensitivity.

DNA双链断裂时转录活性的活细胞成像

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far ...