作者承认口腔医学院的在匹兹堡，健康奖K01 AR062598的国家机构，并授予P30 DE030740的大学的财政支持。作者还感谢金章博士为ICUE1质粒，韦恩Rasband开发的ImageJ和迈克尔Cammer为ImageJ的宏。

The authors have no conflicts to declare.

这项工作表明FRET成像如何可用于分析细胞信号在三维水凝胶。虽然现有的研究已经表明与水凝胶38接种在水凝胶基质35-37，细胞外相互作用的细胞的FRET成像和截留在水凝胶39-41的分子，这是第一次出版物描述嵌入细胞FRET成像基于细胞的分析3D水凝胶。从2D转换FRET成像3D时，工作解决了一些执行问题。首先，需要高数值孔径目​​标FRET成像收集到足够的发射信号，但它们限制景深。这里的允许细胞通过离心来增加细胞数目的焦平面玻璃附近以补偿此稍向定居。第二，三维水凝胶支架可变得复杂分析成像期间横跨视场漂移。水凝胶在这里被结合到COVerslip使得它们保持固定在整个实验。因为水凝胶可能会阻碍细胞的可视化三，3D FRET适当的水凝胶组合物的选择是至关重要的。 PC-凝胶和TR-凝胶这里透明的水凝胶被使用。然而，收集用离心分离使细胞在盖玻片附近的焦平面可以用于图像的细胞在水凝胶具有较高的水凝胶的衍射仪选择取决于微环境条件必须模拟，其可在评论中找到对凝胶42 。第四，一个备用计算此处使用的单链ICUE1探针的FRET比率（ 即 ，E QE = QE / CAEM），以尽量减少分析所需的图像的信道数，从而减少光漂白。每个细胞的平均FRET比率被计算为平均每像素的比率的小区内，以尽量减少实际的FRET比率的低估。最后，一​​个线性比例分析和装置计算单链二元探针的激活的级分进行说明。 有使用广角镜进行成功的FRET实验的几个关键方面。对于硬件，相机必须足够敏感来解决小信号变化，而使新科学的CMOS摄像机和电子倍增CCD比传统的CCD更适合。到最好的分析的FRET比率的空间分布，相机必须至少具有物镜（奈奎斯特准则）的点扩散函数的空间分辨率的两倍。这使得双视图系统不太适合的任务。更关键的是，选择用于给定FRET对适当的曝光和图像采集速率以便最小化的荧光团的光漂白。降低采集速率被推荐为实验的持续时间增加。使用快速滤光轮的提高采集速率和视场进行分析，同时避免了数ING与双视图和炮塔滤镜立方体图像配准的问题。不均匀的照明领域的复杂化，从样品自发荧光和摄像头的系统噪声产生的背景荧光校正。一些水凝胶，特别是那些含有胶原蛋白是高度自体荧光43,44。该FRET比率被低估没有背景校正。在这里，我们采用依赖于它可以经常与落射荧光照明在图像上（ 图3B，步骤7.2）的中心被配置在相对 ​​平坦的照明场的简单的背景校正方法。因此，这种方法限制了感兴趣的那些该照明领域内的细胞。这里所描述的背景校正不考虑在读取二进制探针波长蜂窝自发荧光。在这样的情况下，未标记的细胞（无探针转染）应在相同的条件和背景减去下进行成像。 A M可以使用标记和未标记的细胞的九和选定为背景的ROI的未标记细胞。这为在整个图像领域细胞分析替代方法包括使用遮光膜的，多背景的投资回报，或相同的样品不含细胞和协议可应要求提供。 具体到3D FRET成像，探测响应是向水凝胶渗透性分析物（ 图6）高度敏感。因此相同的水凝胶区域横跨实验处理相比，优选在几何对称点如邻近如这里使用的支架中心。可替代地，微流体腔室/生物反应器可用于迅速而均匀地灌注与分析物溶液45的水凝胶。音柱信号可能不会被检测到（信噪比过低）时，水凝胶的自发荧光过高;应使用的较薄的水凝胶或不同探针（不同的荧光团）。在光交联水凝胶相对于3D FRET成像，建议使用最小辐射曝光和探针的荧光团的激发光谱外的波长。 LAP可以与可见光源在405nm被用来进一步减少潜在的细胞损伤31,46。但是，建议使用365纳米的照射LAP因为这减少了探测器漂白。 365nm处在于在CFP施主激发光谱的尾端，而405nm处在于在峰值激发。可替代地，探针的表达可以被允许一天恢复。二进制探针的使用最小化灵敏度的问题，以在表达水平，并在供体和受体荧光团的分子扩散的差异。非二进制探针可以被使用，但具有SE代替QE成像和附加校正激发和发射光谱的光谱渗出至（见下文）。此外，低商业可用性和设计的FRET探针的复杂性可能是一个障碍。对于成功的实验的最关键因素仍然是荧光信号的适当的修正和分析。 FRET的比率的替代计算被用于除了光漂白的最小化几个其他原因。对于二进制单链探针时，常见的比例是根据捐助者的激励（致敏的排放，SE），以供体发射下捐助激励（淬火排放，QE）， 即受体发射，FRET比= SE / QE 25,47,48。 SE / QE是非线性相对于FRET效率21,22,47变化。即，该比例具有与探针（EI）的固有的FRET效率的指数的非线性关系（Donius，AE，Taboas，JM 未发表的作品。（2015）），与荣小于大约15％的比例最线性的。 SE / QE还将低估激活探针（FAP， 即，探针结合分析物的级分）的分数。 Therefo再次，SE / QE的分析需要繁琐的平衡剂量反应研究，曲线拟合。幸运的是，SE或QE到受体发射下受体激发的比例（AEM）是相对于Ei和FAP， 即直链的，所述的FRET比率SE / AEM和QE / AEM。 SE / AEM通常用于二进制探针，只需要结束点校准（在无探针的活性和全探针活性），但基底细胞信号使这个困难。为了消除对端点校准的需要，SE能对供体和受体的激发和发射光谱的光谱重叠（光谱泄放通）和用于将合并的探针的荧光团和显微镜的量子产率和光谱灵敏度（QS）进行校正（CSE）成像系统。基于CSE校正后的SE的FRET比率限定的图像， 即，E SE = CSE / AEM的每个像素内的探针的观察FRET效率。商业和免费软件可供SE纠正这些影响和读者可参考陈和2006 Periasamy的工作的方法50的详细说明。但是，计算êSE要求每个时间点（SE，QE，AEM）三个图像通道捕获。因此，在此工作中，我们也使用一个替代的FRET比E QE = 1 - QE / CAEM，其中只需要两个图像通道的捕获（QE和AEM）。从这些渠道计算ËQE只需要修正为AEM QS（CAEM = AEM x质量得分）。 QS = DEM的/ AEM利用从一个校准样品，其中，数字高程模型是根据供体激发的供体发射与受体漂白两个图像容易地估计。 FAP的在任何时刻可以用E SE或E QE比较探针的荣来计算。 最终，FRET比选择（E SE = CSE / AEM 与 ËQE = QE /航空气象学委员会）应基于考虑探头类型，并与最佳的信号通道。既是pproaches包括如上所述以考虑随时间的自体荧光的改变每帧的图像的背景噪声的校正。他们不承担任何AEM激发光重叠到民主党激发光谱，这往往是由于探头设计和显微镜的过滤器配置的情况下。单链探针可以使用和选择是基于最好的探测信号（亮度）到相机噪声，并就SE和QE通道背景信号。对于ICUE1探头和这里使用的实验条件（细胞和水凝胶型），SE比QE更强的信号。双链探头需要采用E SE由于供体和受体荧光团非等摩尔分布。对于在结合分析物如ICUE1在FRET降低，FRET效率可以"倒"呈现在结合分析物的积极信号的变化，因为我们在这里做的，有E SE = 1探测器- CSE / AEM或E QE = QE / （AEM x质量得分）。 日分析ËFAP是对FRET比率适当修正，并荣的估计敏感。它可以与用于QS上的相同的校准样品和常规的端点来估计FRET效率测定为荣= 1-（QE / DEM）（QE图像后跟受体漂白和DEM的图像）28，但必须小心，以尽量减少捐助意外漂白。我们描述那里调整QS的民主党基于AEM估计被取代，修改后的版本，即 ，EI = 1 - （QE /（AEM x质量得分））。交替，可以使用荣= CSE / AEM。在活细胞荣估算，要求所有探头被驱动到全FRET。这是在实践中难以为探针，如ICUE1即在结合分析物中的FRET减少来实现。使用细胞裂解物和纯化的分析物荣的体外基于计算是最好的，但这种工作的范围之外。在这里，我们建议使用2&#39;，5&#39;-二脱氧腺苷，以减少在细胞内cAMP。然而，一个相对的FAP可be。使用从信令的基线水平得出的EI估计计算。由于这些原因，研究最经常报告基于端点校准，其中加入激动剂之前，信令基线是下界和信号并称饱和信令的第二激动剂后的FRET比率（如这里完成）或相对FAP在上界。福斯克林常被用作对照激动剂饱和的cAMP信号。交替，可以使用cAMP类似物如8- Bromoadenosine 3&#39;，5&#39;-环一磷酸。在的研究，以确定在试验性治疗中的信号传导途径的潜在变化的完成使用阳性对照建议。 每个细胞的平均信号传导应答的计算需要考虑的几个因素。首先，将平均FRET比率应计算的平均值的比率在每个像素的小区内， 即 ，（Σ（QE/cAem））/（像素数），而不是岭平均QE和AEM在细胞， 即 （ΣQE）/（ΣcAem）的蒂奥。尽管后者不需要小心掩蔽作为背景噪音低，它严重低估了真实平均FRET比率。然而，像素比值需要浮点运算和单元面积来定义的ROI和避免包含从细胞外的背景噪声产生的寄生比的仔细二进制掩蔽。整个小区区域必须量化以避免对细胞形状偏压的结果。掩蔽可能需要重新定义随时间根据在细胞形状和迁移的变化。或者，如果排除标准定义为从QE并且大大低于细胞水平和接近背景水平AEM信号中去除的像素的比率，可以使用的ROI比电池大。所描述的分析方法的细节在此工作的FRET分析中使用无需专门软件/插件的基本步骤。显微镜制造商和其他供应商销售附加模块S代表他们的显微镜软件，它支持自动比例和FRET分析。同样，公共领域的ImageJ软件具有可用于颗粒和FRET分析几个插件，如RiFRET。第二，基于像素的平均的FRET比率低估，因为2D图像使用三维微孔结构的真正的平均比率。二维信号表示沿z轴的平均值。在活细胞中的FRET比率的三维空间分布的计算是不可能的，而不高速三维成像（ 例如 ，使用旋转盘共焦显微镜）。这是二维和三维培养物中的问题，但在3D较大效果更蜂窝结构中存在的平面外。这对定位于细胞结构，例如细胞膜EPAC1探针PM-ICUE探针的极大关注。见Spiering等。 2013年的建议，以修正对比率计算，24电池厚度的影响。 AEM的分布的分析可以用来如果检测探头调节细胞和成像平面的区域聚集。这也将有助于解释FRET比率的空间分布，并消除虚假的结果， 例如 ，识别探针饱和度（ 即，低的AEM区域将具有低探针浓度和可以表现出探针饱和和高的FRET比率）。第三，不同的FRET基准水平可以指示在转染效率的差，探测实验组之间的表达，或基底信令。 "相关性分析"（步骤10.1.1）的帮助，如果存在删除漂在FRET比率随着时间的推移。它促进的样品之间的反应的相对大小的比较，但阻碍比较响应为参考样品（对照区是一个平线）的时间过程。 "绝对分析"（步骤10.1.2）便于在样品反应速度的比较，但因为在每行由一个dissim缩放妨碍反应的幅度的比较ILAR不变。研究经常使用在基线上的线性回归以校正在FRET比率随时间漂移。 所描述的3D FRET方法是制造和执行小区载货三维水凝胶，其可以被应用于其它探针和成像方式的时间推移成像有用的和实际的方法。除了单链二元探针其它FRET系统将需要基于强度的信号的更复杂的校正，如上所述。另外，FRET（FLIM-FRET）的荧光寿命成像可用于通过探头供体发射寿命的变化来计算FRET效率。不像基于强度的FRET，FLIM-FRET是不敏感的背景噪声，光谱渗出通过，量子效率，和检测器的光谱灵敏度2。然而，FLIM系统是昂贵的，复杂的，和不常见的，并与单指数衰变的荧光团和无FLIM的径流49工作最好。所描述的方法也可以与莫用于再先进的显微镜平台（ 例如 ，FRET的TIRF，荧光各向异性，谱相关FRET）。应用这种方法对三维成像高速共聚焦和多光子显微镜将促进信令响应的亚细胞分布的分析和增加信令分析的准确度。这种3D FRET方法将使模拟3D微环境细胞的高级细胞生物学研究。因此，它可以容易地应用到药理学和再生医学的需求，包括研究细胞间信号传导和筛选在改造的水凝胶基microtissues药物反应。

细胞信号既复杂又衍生了众多领域，包括药理学，组织工程和再生医学。有用的研究性工具需要进一步我们的细胞生物学的理解，并发展组织再生的最佳材料。荧光共振能量转移（FRET）成像是一个重要的工具，使受体激活，分子构象，并在活细胞中的超分子相互作用（ 例如 ，蛋白质/ DNA复合）的分析。 FRET是一种荧光团1之间的非辐射能量转移的。它有一个效率是成反比的供体荧光团和受体荧光团之间的距离的第六功率。因此，它可以提供有关的空间距离在纳米尺度两个不同分子之间或跨越一个分子的区域的信息（通常为1 - 10纳米）2。供体和受体荧光团可以是不同的Molecule类型（杂-FRET），其中该供体具有更高的能量发射（较短波长），或者相同类型的（同型FRET）。 FRET成像可以在固定的或活细胞中进行，但在一般的固定细胞用于分子间的相互作用的成像在高空间分辨率时高速共聚焦系统不可用。活细胞用于研究的相互作用和过程被抑制或通过定影改变，如实时细胞内信号响应3。 雇用活细胞研究FRET显像使用二维（2D）细胞培养物中，因为简单和低的背景（无从平面外的细胞的发射）的一部分。然而，2D培养物也相比于生理微环境和三维（3D）培养4,5-改变众多细胞过程。例如，细胞和细胞外基质相互作用天然细胞被彻底改变在二维培养导致EASILY观察细胞形态和极性6的变化。膜微（ 例如 ，小窝和脂筏）和受体信号强烈的文化环境，因为它们结合细胞骨架7,8和细胞形态和细胞骨架结构在很大程度上受到空间的养殖环境调控9上调，部分。机械传导不仅由3D矩阵的影响，但由更复杂的二级载荷条件在3D环境主义及其相比2D培养物10。最后，三维矩阵的渗透率小于培养基，一般具有降低的扩散和增加的信号传导分子相比2D培养细胞的结合。与3D培养人能够创建并观察一个微环境更类似于生理相对于粘合剂，化学和机械的线索。细胞与细胞的相互作用可促进和粘接性能能够控制瓦特第i个的结构中，组合物，和3D材料11-13的体系结构（图案形成）。该材料的机械性能同样可以通过组成和结构14,15量身定做。培养细胞在水凝胶，因此允许FRET上，否则将去分化的原代细胞或使用常规技术在表型变化， 例如研究中，从关节软骨细胞。因此，需要一种方法，以使在现场的3D培养物的FRET测定。 水凝胶的支架是理想的基于三维的FRET的研究，因为它们可以被制成光学透明，并适合控制微环境，并提供蜂窝线索。从天然聚合物制成的水凝胶已经在细胞培养中使用了几十年，包括明胶和纤维蛋白16。在细胞微环境更大的控制可以与这些聚合物的化学改性，并与使用合成聚合物17,18来实现。 HYDRogel机械刚度和渗透性可以通过改变它们的聚合物组合物进行控制和网眼尺寸（聚合物和交联密度）19,20。此外，水凝胶可以通过生长因子和细胞的配体的结合进行的生物活性。由于这些可能性，水凝胶生物传感，药物输送和组织工程应用的发展研究21一 ​​个非常活跃的领域。水凝胶的三维打印也正在开发微型组织制造和-organs 15。因此，在水凝胶的细胞FRET成像不仅作为近似生理细胞行为的手段是有用的，但也可作为工具来研究和优化工程化组织的生长。 二进制比率的FRET探针是在研究细胞信号传导非常有用的，可以在水凝胶培养物被利用。对于荧光公布的部分名单，FRET探头，看到细胞迁移联合会网站<sup&gt; 22。最常见的探针类型包含由一个识别元件（多个）（分析物敏感区域）关联或连​​接的两个不同的荧光团。这些区域经历构象变化，联系或解离在检测分析物（ 例如 ，离子或分子，该区域的酶裂解的结合），该改变荧光团，因此FRET幅度（或高或低）之间的距离23。一个不太常见的但却很有用的探头类型依赖于两个荧光基团的光谱重叠，从而改变FRET幅度分析物敏感的变化。 "单链"比例式传感器利用荧光团对上的单分子相连。 "双连锁"探测器利用在不同的分子荧光团对，但他们的表现可以在相同的表达盒24下耦合。不同于单一的荧光表达研究（ 如 ，荧光融合蛋白），研究与比率FRET探针不需要双重转染，以控制转染效率或细胞生存力。最低门槛（浓度不敏感）23,25以上时，表示比率FRET探针是相对不敏感的转染效率。它们是不敏感的激发源的波动和其它细胞内环境因素（ 例如 ，pH值，离子），只要这两个荧光团也同样敏感。此外，他们的反应是不受转录延迟，不像荧光和生物发光启动子-报道构建26,27。 成比例的FRET探针轻松，频繁地在传统的宽视场落射荧光显微镜系统中采用。宽视场显微镜优于由于超过系统成本，荧光漂白，并以足够的时间分辨率信号改变捕获的关注线扫描共聚焦系统为活细胞成像。另外，单电池肛门ysis在一个人口众多的细胞是可能的机动阶段和图像采集了多视野。广角镜需要异质FRET探针信号的强度基础分析。虽然强度分析不需要像其他的FRET方法复杂的硬件，更采集后工作是需要以校正荧光行为和显微镜/成像系统配置28的影响。荧光团的拍摄发光强度的激发能量的函数，荧光量子产率，并且将合并的过滤器和相机/检测器的光谱灵敏度和线性度2。这些可以是不同的供体和受体，并且需要进行定量分析的校准。此外，该受体发射的成像由荧光团的光谱重叠的影响（受体的激发供体激发光和渗出通过供体发射的到受体发射光谱）2。4单链"探针内部归因为它们的荧光团等摩尔在纳米尺度，而荧光团"双链"探针可以在不同的化学计量存在整个电池25,28如果荧光团。"单链"探针是相似亮度（消光系数和量子产率的功能），非线性检测器的灵敏度的效果是对于背景荧光和耦合量子收率/检测器的灵敏度，需要23小并且仅校正的。因此，"单链"比例FRET探针是最容易在广角镜24实现。 本文介绍一种简单的技术来执行使用传统的宽视场啶显微镜在三维水凝胶文化的活细胞成像FRET。它可以通过已经开展二维FRET实验，以及有意间实验室的实验室很容易地通过在microtissues细胞信号。在这里，我们证明用环磷酸腺苷（cAMP）的光交联内活软骨细胞水平（PC-凝胶，聚乙二醇二甲基丙烯酸酯）和温敏（TR-凝胶，基质胶）水凝胶的FRET成像基于测量的方法。进行比例FRET探针是基于EPAC1（ICUE1）来检测响应佛司可林，对腺苷酸环化酶的化学激动剂其催化ATP向cAMP的转化cAMP水平利用。的cAMP是主要的第二信使介导的G蛋白偶联受体信号之一，并激活各种因素，包括由腺苷（EPACs）直接激活下游交换蛋白。荧光团移动分开时cAMP结合，导致在FRET的减少的EPAC域。这里所描述的是水凝胶材料的在三维水凝胶的细胞的制备中，细胞转染与ICUE1探针和嵌入。三维FRET实验程序和必要的图像分析以评估每个细胞活性探头解释。我们还讨论了在2D和3D用广角镜FRET分析的内在局限性。这里提出的技术是在现有的二维方法的改进，因为它使在一个更生理上下文分析和需要更少的图像校正和校准。极大效用在于，可用于许多透明材料，同时细胞和转染的偏好可以维持这一事实。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="127">
			<td height="127" style="height:127px;width:216px;">Polydimethylsiloxane (PDMS) [Sylgard 184 Elastomer Kit]</td>
			<td style="width:71px;">Fisher Scientific (Ellsworth Adhesives)</td>
			<td style="width:119px;">NC0162601</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:216px;">Polyethylene glycol dimethacrylate (PEGDM)</td>
			<td style="width:71px;">Prepared according to Lin-Gibson with PEG from Sigma Aldrich</td>
			<td style="width:119px;">95904</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">3-(Trimethoxysilyl)propyl methacrylate</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">M6514</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">3-glycidoxypropyltrimethoxysilane</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">440167</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dimethyl Phenylphosphonite</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">149470-5g</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">2,4,6-trimethylbenzoyl Chloride</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">AC244280100</td>
			<td style="width:167px;">Reagents</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">2-Butanone</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">AC149670010</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Lithium Bromide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">AC199871000</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Isopropanol</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">190764</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sigmacote (siliconizing reagent)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">SL2</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Toluene&#160;</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">AC36441-0010</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH3025601</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Opti-MEM Reduced Serum Medium, no phenol red (phenol- and serum-free medium)</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">11058-021</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">FuGENE HD Transfection Reagent (transfection reagent)</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">E2311</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cellstripper (cell dissociation solution)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">25-056-CI</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Growth Factor Reduced Matrigel, Phenol Free</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">356231</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Forskolin</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">F6886</td>
			<td style="width:167px;">Reagents</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipette Tips (10,200,1000 &#956;l)&#160;</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">02-707-474, 02-707-478, 02-707-480</td>
			<td style="width:167px;">Disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Powder Free Examination Gloves</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">19-149-863B</td>
			<td style="width:167px;">Disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Aluminum foil</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">01-213-100</td>
			<td style="width:167px;">Disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Biopsy punch (3 mm)</td>
			<td style="width:71px;">Miltex</td>
			<td style="width:119px;">3332</td>
			<td style="width:167px;">Disposable lab equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass coverslips</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12-544C</td>
			<td style="width:167px;">Disposable lab equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Precoated glass coverslips (epoxysilane)</td>
			<td style="width:71px;">Arrayit</td>
			<td style="width:119px;">CCSL</td>
			<td style="width:167px;">Disposable lab equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass slide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12550D</td>
			<td style="width:167px;">Disposable lab equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile vials (15 mL, 50 ml conical)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-959-70C, 14-959-49A</td>
			<td style="width:167px;">Disposable lab equipment</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell culture dish (6-well)</td>
			<td style="width:71px;">Falcon</td>
			<td style="width:119px;">351146</td>
			<td style="width:167px;">Disposable lab equipment</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Epifluorescent Microscope with motorized stage</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">MEA53100 (Eclipse TiE Inverted)</td>
			<td style="width:167px;">Large/non-disposable lab equipment</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Adjustable Specimen Holder for XY Stage</td>
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			<td style="width:119px;">77011393</td>
			<td style="width:167px;">Non-disposable lab equipment</td>
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			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">MRD01605</td>
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			<td height="106" style="height:106px;width:216px;">CFP/YFP light source and excitation / emission filter holders (Lambda light source &#38; wheel, Lambda emission filter system)</td>
			<td style="width:71px;">Nikon (Sutter Instrument reseller)</td>
			<td style="width:119px;">77016231, 77016088</td>
			<td style="width:167px;">Non-disposable lab equipment</td>
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		<tr height="85">
			<td height="85" style="height:85px;width:216px;">CYF/YFP filter set</td>
			<td style="width:71px;">Nikon (Chroma Technology Corp)</td>
			<td style="width:119px;">77014928</td>
			<td style="width:167px;">Non-disposable lab equipment</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:216px;">Camera (ORCA Flash 4.0 v1)</td>
			<td style="width:71px;">Nikon (Hamamatsu reseller)</td>
			<td style="width:119px;">77054076</td>
			<td style="width:167px;">Non-disposable lab-equipment</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">NIS-Elements 40.20.02 microscope software)</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">MQS31100&#160;</td>
			<td style="width:167px;">Non-disposable lab equipment</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Prism (spreadsheet and graphing software)</td>
			<td style="width:71px;">GraphPad Software, Inc</td>
			<td style="width:119px;">Version 6</td>
			<td style="width:167px;">Non-disposable lab equipment</td>
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		<tr height="85">
			<td height="85" style="height:85px;width:216px;">OmniCure S1000 UV Spot Curing Lamp System with 365nm filter or other light source (&#955;=365 or 405)</td>
			<td style="width:71px;">OmniCure</td>
			<td style="width:119px;">S1000</td>
			<td style="width:167px;">Large/non-disposable lab equipment</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Pipette Aid&#160;</td>
			<td style="width:71px;">Drummond Scientific Co.&#160;</td>
			<td style="width:119px;">DP-100</td>
			<td style="width:167px;">Non-disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Hemacytometer</td>
			<td style="width:71px;">Hausser Scientific</td>
			<td style="width:119px;">Reichert Bright-Line 1475</td>
			<td style="width:167px;">Non-disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Vacuum desiccator chamber/degasser&#160;</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Large/non-disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Tissue Culture Hood&#160;</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Large/non-disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Chemical Fume Hood&#160;</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Large/non-disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Oven</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Large/non-disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Spatula</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Non-disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Beaker</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Non-disposable lab equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Incubator&#160;</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Large/non-disposable lab equipment</td>
		</tr>
	</tbody>
</table>

fret imaging in three-dimensional hydrogels

荧光共振能量转移（FRET）的成像是在实时检查细胞生物学的有力工具。利用FRET研究通常采用的两维（2D）培养，其不模仿三维（3D）细胞微环境。使用3D凝胶环境内的细胞的常规广角荧光显微镜进行骤冷发射FRET摄像用方法。这里描述了比例FRET探针产生在探头激活范围线性比例的分析方法。细胞内环磷酸腺苷（cAMP）水平的测定证明在根据福斯克林刺激软骨细胞用探针为EPAC1活化（ICUE1）和检测cAMP的信号依赖于水凝胶材料的类型，在此光致水凝胶（PC-凝胶差异的能力，聚乙二醇二甲基）和一个温敏水凝胶（TR-凝胶）。与2D FRET方法相比，这种方法需要很少的额外工作。已经利用FRET成像2D实验室可以很容易地采用这种方法在3D微环境进行细胞学研究。它可以进一步工程化三维microtissues应用于高通量药物筛选。此外，它与其他形式的FRET成像，诸如各向异性测量和荧光寿命成像（FLIM），并与使用共聚焦调制照明先进显微术平台，脉冲，或兼容。

如上所述制备所有水凝胶前体，细胞和铸模。小区载货前体溶液流延在PDMS模具，然后凝胶化之前离心/光交联固定化细胞的盖玻片附近并促进FRET成像分析（ 图1）。附带盖玻片水凝胶置于准备PDMS成像井显微成像（ 图2）内。光学配置和图像采集参数然后设置为如图3A所示。为CFP-YFP荧光团对所有四个FRET相关图像通道的捕获证实（参见"光学CONF"。在图3中），因为需要对非二进制探针。然而，在这项工作只有两个图像都需要对单链二元ICUE1探头，QE ="CFP CFP"和AEM ="YFP YFP"（激发发射）。多视场（XY位置多个）被选择，其中几个细胞的均匀的照明区域（ 图3B）内居住。时间的推移收购完成后，将探头信号导出数据。感兴趣区为信道信号的背景校正和细胞分析采用阈值AEM图像从实验（ 图4）的开始被指定。从细胞池因为这些表达中选出足够的细胞（不是太暗淡），但不能太高（太亮）探针（ 图4B）。在每个采集每个象素的QE和SE的FRET比率使用背景相减图像通道的浮点除法计算。每个细胞的平均FRET比率（即每ROI）计算归一化到之前刺激基线信号（ 即，来自所有时间点中减去基线回归），并用误差棒为一体的标准误差作图平均（SEM）。无论是QE和基于CSE FRET比德TECT下福斯克林刺激（100纳米， 图5）软骨细胞的cAMP增加的活性。在QE FRET比是比CSE FRET比背景校正更敏感，因为QE信号由于嵌入水凝胶内的cAMP软骨细胞活性的低基础水平较低。这两种水凝胶类型的（TR-凝胶和PC-凝胶）显示在FRET比率随时间增加（ 图6），这表明在加入毛喉素的增加cAMP的信号传输响应。正如QE FRET比表示，在PC-凝胶水凝胶软骨细胞显示cAMP的变化较大（如图所示）和cAMP（E QE更高的基础水平= 0.19 PC-凝胶与 ËQE = 0.09 TR-凝胶，在基线减去图中未示出）。 <img alt="图1" src="/files/ftp_upload/54135/54135fig1.jpg" /> 图1: 在光交水凝胶（PC-凝胶）细胞分布 </STRONG&gt; 答:细胞载货水凝胶前体离心在盖玻片附近焦平面最大化细胞的数目。 B:这增加了视场的细胞数，并从平面外的细胞减少背景信号。 （比例尺= 200微米） <a href="https://www.jove.com/files/ftp_upload/54135/54135fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54135/54135fig2.jpg" /> 图2:水凝胶在PDMS菜 水凝胶放在盖玻片基地显微成像和分析物刺激一个高大的PDMS良好（10倍量水凝胶）内。在PC-凝胶水凝胶是透明和结合到盖玻片，使它们保持在适当位置在整个测定法。将PDMS孔可以增大以包含比使用除了在更宽的活检穿孔，水凝胶体积的10倍多平台hicker PDMS表。 （比例尺= 2mm）的<a href="https://www.jove.com/files/ftp_upload/54135/54135fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54135/54135fig3.jpg" /> 图3: 采集配置FRET成像 答:本次收购在多个位置配置了图像采集每7秒。对于QE FRET比例计算，只需要两个图像通道的下此处使用的ICUE1探头（二进制单链探针），QE ="CFP CFP"和AEM ="YFP YFP"（激发发射）"光CONF。"在显微镜软件的λ标签栏。 A:视场（"XY"位置）被选中，其中几个细胞均匀的照明区域内居住。图像下面的图显示沿着蓝色的光照强度箭头线遍历通过FOV的中心。 <a href="https://www.jove.com/files/ftp_upload/54135/54135fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54135/54135fig4.jpg" /> 图4:FRET成像分析投资回报率的选择 答:利息率（ROI）游离细胞的一个区域选择纠正每个通道的背景信号。无细胞的水凝胶的图像可以替代地用于背景减除如果细胞密度或迁移是高，或者如果当细胞不均匀的照明场中位于阴影掩模不被使用。 B:使用图像阈值与AEM通道的二进制掩蔽被选定单元格的投资回报。选择比细胞大的投资回报率将每个细胞的平均比例FRET计算产生不利影响，由于夹杂背景ñ产生的胞外比率瓦兹。不建议使用每个小区的每个信道的平均蜂窝FRET比率的计算，因为这低估的比率。 （比例尺= 50微米）， <a href="https://www.jove.com/files/ftp_upload/54135/54135fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54135/54135fig5.jpg" /> 图5:FRET比率的温敏凝胶（TR-凝胶）的比较 无论QE和基于SE FRET检测的比率下毛喉素刺激软骨细胞的cAMP增加活动。毛喉素是一种腺苷酸环化酶激动剂诱导cAMP的产生。当ICUE1探针结合的cAMP FRET减小，并且因此QE FRET比（E QE = SQE /（SAEM x质量得分））增加。相反，SE FRET比率下降，因此绘制为E SE = 1 - CSE / SAEM。在水凝胶嵌入式软骨细胞，在QE˚FRET比率是背景校正比SE FRET比率由于QE信号为低更为敏感，由于低的基础cAMP的活性。误差棒= SEM，N = 25。 <a href="https://www.jove.com/files/ftp_upload/54135/54135fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54135/54135fig6.jpg" /> 图 6: 不同的水凝胶FRET率的比较 在这两种类型的水凝胶的软骨细胞回应毛喉素刺激这表现在增加QE FRET比例随时间增加的cAMP活动。水凝胶型的影响通过多个作用，包括在渗透性福斯克林和基底细胞cAMP活性的差异的细胞反应（E QE = 0.19在PC的凝胶与 ÊQE = 0.09在TR-凝胶，未示出）。误差棒= SEM，正为TR-凝胶= 25，正为PC-凝胶= 15。 <a href="https://www.jove.com/files/ftp_upload/54135/54135fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about FRET Imaging in Three-dimensional Hydrogels at JoVE.com

FRET Imaging in Three-dimensional Hydrogels

荧光共振能量转移（FRET）成像是用于实时细胞生物学的研究的强有力的工具。这里提出了在使用常规荧光显微镜生理三维（3D）水凝胶的微环境FRET显像细胞的方法。被描述为比例FRET探针产生在激活范围线性比例的分析。

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 ...

fret-imaging-in-three-dimensional-hydrogels

Research

JoVE Journal

Biology

13.0K 次观看.  University of Pittsburgh. 荧光共振能量转移（FRET）成像是用于实时细胞生物学的研究的强有力的工具。这里提出了在使用常规荧光显微镜生理三维（3D）水凝胶的微环境FRET显像细胞的方法。被描述为比例FRET探针产生在激活范围线性比例的分析。 

视频: FRET Imaging in Three-dimensional Hydrogels

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

基于芯片的三维细胞培养，在灌注微生物反应器

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

科研

生物学

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 ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

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 ...

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 ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

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 ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

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 ...

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. 
This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. 
Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.

使用连续截面电子显微镜对肝细胞中组织间接触位点进行三维表征

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is ...