这项工作的经费由荷兰科学研究组织（NWO）以中央公积金的VIDI授权号H64.09.016。 CPF是感激医生Silvina A. Fratantoni她的批评意见，并在最后的手稿更正。 GMRDL / EMMM公司受荷兰技术基金会STW（项目12151和11350），这是苏国的一部分，这部分是由经济事务部资助支持。我们感谢尼康仪器Europe BV公司的凯瑟琳·基茨和彼得·德​​兰特的援助和支持。被授予荷兰科学研究组织（补助金820.02.006）支持HX是支持艺术和科学的荷兰皇家科学院（补助11CDP10）和WT。

所有涉及动物实验的程序进行了优化，以减少动物的痛苦，并获得证监会核准的动物实验，阿姆斯特丹大学，赤纬协议＃DED204和DED250。 1，准备盖玻片<ol><li>切下来的盖玻片为15毫米×15毫米用碳化物或金刚石划片的尺寸，以使它们放入一个12孔板的孔中。 </li><li>而在通风橱内工作时，通过充分浸没的盖玻片在浓硝酸溶液（70％重量/重量）在玻璃容器中开始盖玻片涂层。为了确保均匀分布的抖动，然后孵育至少4小时的。它可以重复使用大约2倍的浓硝酸溶液，但它可以通过暴露于光散色。 </li><li>除去浓硝酸溶液和蒸馏水洗涤盖玻片四倍30分钟，每次洗涤后小心摇动。 </li><li>小心取出水。 注：接下来的三个步骤是在无菌的层流柜进行。 </li><li>浸泡盖玻片在96％的乙醇。从乙醇溶液中取出盖玻片，让他们干。 </li><li>火焰盖玻片，并把它们放在一个玻璃容器。用细镊子处理的盖玻片（杜蒙风格工作日钳为例）。 </li><li>覆盖容器用铝箔和烘烤在12〜16小时的干热烘箱中于180℃下烤盖玻片可存放在室温下长达1个月，如果盖紧。 </li></ol> 2，涂层盖玻片盖玻片涂层过程有利于神经细胞附着到玻璃表面和树突分支14。 <ol><li>在无菌层流罩，用消毒小镊子把个人盖玻片在单井12孔板的。 </li><li>加入500μl的聚-L-赖氨酸溶液（或足量的淹没盖玻片）。 </li><li>包裹吨他板铝箔，以防止蒸发，让它在室温下过夜。 </li><li>开始培养之前，小心地吸出聚-L-赖氨酸溶液在无菌的层流罩。 </li><li>各用1ml无菌水洗涤两次以及洗，同时防止它们干燥。 </li><li>吸出的水完全，加入1 ml电镀液中，并留在组织培养孵化器的盖玻片，直到准备培养板上的细胞。建议在24小时内板上的细胞。 </li></ol> 3，拆卸的大脑从E16-E19大鼠胚胎<ol><li>通过加热在干燥灭菌过夜或用70％乙醇洗涤它们消毒手术器械。彻底干燥，如果乙醇使用。 </li><li>准备几个30毫米菜用1X HBSS缓冲液，并保持他们在冰上。 </li><li>安乐死大鼠坝腹膜内注射Euthasol的（160毫克/千克Euthasol在±0.4毫升的体积）。 </li><li>核实没有反射的。 </li><li>喷雾大坝的腹部用70％乙醇。 </li><li>使沿腹部切口，取出子宫。 </li><li>从子宫中取出胚胎，并将它们放在一个直径100毫米的培养皿。 </li><li>除去大剪刀胚胎的头部，然后将头部中含有冷的1×HBSS缓冲一个新的培养皿。 注意：从这里在无菌条件下在层流柜中执行的程序到步骤7.1。 </li><li>按住头以及大钳的两侧。 </li><li>用小剪刀，使在头顶部皮肤矢状切，然后横向剥离皮肤下的大钳。 </li><li>使用相同的方法与步骤3.10移除头骨。使矢状切开始在尾端;轻轻地打开颅骨而不损伤脑组织。折叠头骨的两半了横向露出大脑。 </li><li>用钝铲，舀出大脑，并将其放入新鲜的冷的1×HBSS。 </li></ol&gt; 海马的4。解剖这是非常重要的是，剥离，尽可能快地完成在无菌条件下，以确保细胞活力。保持样品寒冷的冰。 <ol><li>取下并扔掉小脑与细剪刀。 </li><li>通过使沿中线矢状切分离大脑的两个半球。 </li><li>就拿每个半球，并放置在两个含有新鲜冷的1×HBSS一个新的30毫米的菜。 </li><li>将每个半球，使得颞叶朝向培养皿的底部。 注：从这里开始，建议使用解剖显微镜。 </li><li>轻轻地用小镊子持脑并用另一双钳取出脑。离开半球的其余部分完好无损含有皮质和海马。 </li><li>翻身的组织，使海马现面向培养皿的底部。 </li><li>轻轻握住半球用p花边用细镊子。使用另一种细镊子，小心地，轻轻取出脑膜。它是更容易开始在嗅球。请小心，以免损坏海马。 </li><li>东方的组织，使海马现在朝上。海马现在可通过其特征的C形结构可以看出。 </li><li>用细镊子，解剖出海马。它收集在含有新鲜冷的1×HBSS一个新的30毫米的菜。 </li></ol> 5，细胞分离和电镀<ol><li>海马计数的总数，然后切成小块。 </li><li>收集的碎片，在含有3毫升的1X HBSS一个15毫升的离心管中。 </li><li>离心300×g离心5分钟，小心取出上清液。 </li><li>加入6微升每海马胰蛋白酶。 </li><li>孵育最大20分钟，在37°C。 3分钟后，漩涡。 </li><li>洗2次用5ml新鲜冷的1×HBSS，并弃去上清液。 </li><li>塞科后ND洗净，加1.5毫升电镀液中，预热至37°C。在电镀介质中的血清会失活胰蛋白酶的活性。 </li><li>慢慢磨碎30X与火抛光的巴斯德吸管，直到组织的所有部分都均匀分散成单个细胞。避免起泡。 </li><li>加入5 ml电镀的介质预加热到37℃。 </li><li>采用台盼蓝活体染色计数细胞。 </li><li>播种每孔50000个细胞的12孔板中的1ml电镀培养基中，在第2（总体积2毫升）溶液制备。 </li><li>轻轻摇动板均匀分布的细胞。 </li><li>在37℃，5％CO 2。后2-3天，用含有10μMFUDR培养基替换一半的电镀培养基（0.5ml）中。 注：树突棘成像电镀后16-17天进行（16-17天在体外 ，DIV）。 </li></ol> 6。大鼠海马神经元主要使用转染脂质体<ol><li>准备表达GFP和培养液（10毫升Neurobasal培养基用100微升GLUTAMAX的）质粒DNA。 </li><li>预温孵化中期至37°C。 </li><li>准备DNA混合物（A管）。对于每个盖玻片加1微克的DNA在100μl纯Neurobasal培养基中。轻轻混匀。 </li><li>准备脂质体混合物（B管）。对于每个盖玻片在100μl纯Neurobasal培养基中加入2μl脂质体。轻轻混匀。 </li><li>添加脂质体混合的DNA混合溶液。 </li><li>孵育在层流罩30分钟，在室温下进行。 </li><li>加入1 ml预热的孵化介质在板1。 </li><li>存储板2为5分钟，在37℃，5％CO 2。 </li><li>轻轻地逐滴加入200微升的DNA /脂质体的混合到每个孔中并孵育45分钟，在37℃，5％CO 2。 </li><li>随着使用小镊子提起coversliPS含有神经元和它们浸泡在含有新鲜温暖的Neurobasal培养基3 cm的天线冲洗，将其移动到板2。 </li><li>孵育转染的神经元，在37℃，5％CO 2的48小时。 </li><li>检查转染效率转染后24小时。 </li></ol> 7，安装免疫染色的大鼠海马神经元原发性和为了提高转染细胞荧光强度，进行免疫染色协议，以提高检测的GFP转染后48小时。 <ol><li>准备4％PFA和0.05M的TBS。 </li><li>温暖的4％PFA溶液至37°C。 </li><li>轻轻地从含有盖玻片的孔中吸出培养基。 </li><li>加入500μl温暖的4％PFA仔细，以免损坏树突。 </li><li>在室温下孵育15分钟。 </li><li>用1×HBSS中5分钟洗涤3次。 注：在这一点上的样品可以存储长达3周，在4℃或免疫染色可以立即开始。 </li><li>如果样品储存，洗净用0.05M TBS 3次，每次5分钟。 </li><li>在室温下30分钟的TBS-BSA（1％）溶液进行阻挡。 </li><li>用0.05M TBS 5分钟洗涤3次。 </li><li>添加稀释在温育混合物的初级抗体。 </li><li>孵育板1小时，在室温下进行。 </li><li>此外，孵育过夜，4℃下轻轻摇动。 </li><li>除去第一抗体。 </li><li>用0.05M TBS的3倍5分钟洗净。 注：从这个角度上，保持避光的盖玻片。 </li><li>添加辅助荧光标记的抗体稀释孵育混合物。 </li><li>在室温下孵育2小时。 </li><li>去除二级抗体。 </li><li>用0.05M TBS 5分钟洗涤3次。 </li><li>用1X结核5分钟，洗2次。 </li><li>用细镊子从井中取出盖玻片。 </li><li>干用纸巾任何多余的结核病和安装COVerslips使用的安装介质。 </li><li>用指甲油密封，防止安装介质的蒸发。 </li></ol> 8，树突棘成像使用结构照明显微镜使用的材料中描述的SIM系统树突棘成像具有横向分辨率约为85-110纳米（XY）值和200之间的轴向（Z）的分辨率值 - 250纳米，提供2倍分辨率的提高的一个因素相比，宽视场显微镜。 注意：使用的SIM树突棘成像典型的做法是一步7.22后2天，但可以做长达3个星期后，如果样品被蒙在鼓里和22受控温度下 - 23°C。 <ol><li>转动488纳米的激光，汞灯，级控制器，压电调节器中，卤灯，用于发射光并在PC上，并在“ANDOR为N-SIM”模式启动的SIM软件。 </li><li>清洁100X TIRF对象的ive用95％乙醇3次，并在必要时，用石油醚。 </li><li>使用下面的过滤器设置：520 LP有488分色。 </li><li>把浸油对目标下降。检查有没有气泡中的油滴。移动目标向上直到油接触到样品。 注意：将一个盖过来保护样品不受环境光线和昏暗的灯光在房间里尽可能的阶段。 </li><li>物镜的校正彩色设置为37℃，200微米，以获得PSF的最好的对称性。为了设置正确的领口位置，第一个位置在最佳标称位置的客观环，然后检查一个100纳米磁珠的样本。按照最好的PSF的对称性，稍微改变围绕标称1套环的位置。 </li><li>用于照明，使用3D-SIM光栅（3D 1layer 100X/1.49所有波长）。开始光栅对准的地方所选光栅块插入SIM照明，与100X 1.49目标到位。使用安装在介质100纳米磁珠样品，用浓度，可以让孤立10-15珠子的视角（FOV）的字段。客观修正衣领设置到所需位置后，选择3D-SIM照明和启动软件引导的调整过程。它将运行（5阶段）×（1方向）×（100的z平面）的图像，从其中将重构FOV与珠。通过适当的投资回报率选择单个珠子，覆盖整个珠，包括失焦的模糊的光后，软件将启动一个自动的PSF拟合，它会根据结果调整光栅的位置。 </li><li>检查显微镜的性能，因为重复引起的表的动作和/或温度漂移可能的失准的光栅对准每2周。此外，根据制造商的建议，还检查激光强度和稳定性。 </li><li>用95％乙清洁样品表面anol三次。 注意：对于接下来的步骤见图3的控制面板和内部的SIM软件的正确设置的概述。 </li><li>在SIM卡的软件，选择光学配置眼睛FITC和在炮塔1选择一个空滤块，用于目视检查与白光。 </li><li>设定的对焦速度和XY工作台，以“微调”的行驶速度。 </li><li>打开快门和快速聚焦在样品上。 </li><li>再次关闭快门，并将Z坐标为零。 </li><li>在炮塔1选择绿色的过滤器使用绿色通道目视检查。 </li><li>设置汞灯为最低值的强度。 </li><li>移动到样品的边界小心，确保目标不接触密封剂。 </li><li>打开快门并迅速通过与可能的最低强度的样品进行扫描。 </li><li>当遇到感兴趣的足够明亮的枝晶，围绕感兴趣的片段在视场中，关闭快门。 </li><li>软件设置为光学配置3D-SIM卡488和相机设置到读出方式的EM，获得1 MHz的16位，曝光时间100毫秒，激光功率5％和EM获得200。 注意：请检查绿色滤光片在炮塔2被选中并且炮塔1是空的。 </li><li>检查光栅被设置为“移动”，然后单击现场用激光，并通过摄像头查看示例。 </li><li>激活查找表。 </li><li>如果有必要围绕感兴趣的对象和焦点与对焦速度设置为极精细。 注意：在读出模式EM增益1 MHz的16位，目标强度的好SIM卡的图像是30,000-45,000之间。 </li><li>快速调整相机设置以获得在读出模式EM增益1 MHz的16位30,000-45,000之间的强度值。首次使用： <ol><li>激光功率：0％ - 20％（以上述制备的样品，5％或2.6毫瓦就足够了） </li><li>曝光时间：50毫秒，2秒</li><li>读出模式：EM增益1 MHz的16位</li><li> EM增益：0-300 </li><li>转换增益：1倍 - 5.1倍</li><li>格式直播：无分级</li><li>格式捕获：无分级 注意：这些设置6.3％±1.3％漂白的技术实现的，可接受的最大脱色，这可能显著影响图像质量15的10％限额内。 </li></ol></li><li>单击停止，关闭实时取景。 </li><li>在ND序列面板配置3DŽ堆栈的设置： <ol><li>范围：2微米</li><li>设置尺寸：120纳米</li><li> Z平面</li><li>点击首页位置</li><li>在lambda部分选择的光学配置3D-SIM卡488 </li></ol></li><li>选择3D-SIM作为在N-SIM卡垫的采集模式。 </li><li>运行ND序列采集并保存原始数据。 </li><li>再次选择光学配置眼睛FITC和重复步骤8.15-8.27，直到整个样品已成像</li><li>三维图像重建：在步骤8.23获取的数据可以重建马上或稍后。开始由Z堆栈与重构切片或重建堆栈模式的默认设置重建重建，必要时进行调整。 注：为获得最佳效果，Z堆栈应与所指示的步长和重建中重构堆栈模式。总是通过比较它的原始数据，或者最好是在宽视场图像检查重建图像的有效性。可以调整为在重建过程中的参数是对比度和高频率的噪音抑制。两者影响不同原始图片属性如何在重建过程中考虑进去。在箱子低的调制深度的原始数据时，对比度参数起着重要的作用。如果用户拥有的数据集与一个低信号 - 噪声比，则高频的噪声抑制参数可以影响重建质量本身verely。 </li><li>当成像完成后，中心的XY工作台和移动目标，一路下跌到其静止位置。 </li><li>卸载样品并清洗样品和目标用95％的乙醇。 </li><li>关闭软件，并在PC和关掉所有其它设备。 </li><li>所获取的图像的三维重建和脊柱的分类可以根据使用NeuronStudio软件之前描述的转换的文件，以TIFF格式（参见图4）16以后进行。 </li><li>使用重建下列参数在NeuronStudios软件： <ol><li>体积：体素尺寸：X：0.03微米; Y：0.03微米; Z：0.120微米。 </li><li>枝晶检测：附加比：1.3;最小长度：5微米;离散率：1;重新调整路口：是的。 </li><li>脊柱检测：最低高度：0.2微米;最大高度：5.001微米;最大宽度：3微米;最小粗短的大小：10像素;最小的非粗短大小：5 VOXELS。 </li><li>脊柱分类：颈部比（头颈部比）：1.1;薄比：2.5;蘑菇大小：0.35微米; </li></ol></li><li>轴突顶点形状：用于渲染NeuronStudio参数固体蚀;突起的顶点颜色：按类型;突起的边缘形状：线;突起的边缘颜色：单色;脊柱形状：固体蚀;脊柱颜色：按类型。 注意：在三维重建是它也可以设置音量渲染。预设的阈值被设置为20的“重新生成体绘制”选项活跃。不透明是由“自动亮度”检查设置。 “表面只有&#39;和&#39;使用点预渲染”选项也很活跃</li></ol>

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G., Agard, D. A., Sedat, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I5M:+3D+widefield+light+microscopy+with+better+than+100+nm+axial+resolution.">I5M: 3D widefield light microscopy with better than 100 nm axial resolution.</a> J Microsc. 195, 10-16 (1999).</li><li>Heintzmann, R., Cremer, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laterally+modulated+excitation+microscopy:+improvement+of+resolution+by+using+a+diffraction+grating.">Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating.</a> Proc. SPIE 3568 Optical Biopsies and Microscopic Techniques III. , 185-196 (1999).</li><li>Karadagli&#402;&#225;, D., Wilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+formation+in+structured+illumination+wide-field+fluorescence+microscopy.">Image formation in structured illumination wide-field fluorescence microscopy.</a> Micron. 39, 808-818 (2008).</li><li>Schermelleh, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction+multicolor+imaging+of+the+nuclear+periphery+with+3D+structured+illumination+microscopy.">Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.</a> Science. 320, 1332-1336 (2008).</li><li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+structured-illumination+microscopy:+wide-field+fluorescence+imaging+with+theoretically+unlimited+resolution.">Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.</a> Proc Natl Acad Sci U S A. 102, 13081-13086 (2005).</li><li>Nagerl, U. V., Willig, K. I., Hein, B., Hell, S. W., Bonhoeffer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+dendritic+spines+by+STED+microscopy.">Live-cell imaging of dendritic spines by STED microscopy.</a> Proc Natl Acad Sci U S A. 105, 18982-18987 (2008).</li><li>Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L., Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+video+microscopy+of+live+cells+by+structured+illumination.">Super-resolution video microscopy of live cells by structured illumination.</a> Nat Methods. 6, 339-342 (2009).</li><li>James, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+microcontact+printing+of+micrometer-scale+poly-L-lysine+structures+for+controlled+growth+of+cultured+neurons+on+planar+microelectrode+arrays.">Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays.</a> IEEE Trans Biomed Eng. 47, 17-21 (2000).</li><li>Dumitriu, D., Rodriguez, A., Morrison, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput,+detailed,+cell-specific+neuroanatomy+of+dendritic+spines+using+microinjection+and+confocal+microscopy.">High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy.</a> Nat Protoc. 6, 1391-1411 (2011).</li><li>Fitzsimons, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+the+glucocorticoid+receptor+alters+functional+integration+of+newborn+neurons+in+the+adult+hippocampus+and+impairs+fear-motivated+behavior.">Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior.</a> Mol Psychiatry. , (2012).</li><li>van Hooijdonk, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-mediated+transgene+delivery+to+the+hippocampus+reveals+sub-field+specific+differences+in+expression.">Lentivirus-mediated transgene delivery to the hippocampus reveals sub-field specific differences in expression.</a> BMC Neurosci. , (2009).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1, 2406-2415 (2006).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35, 567-576 (1993).</li><li>Izeddin, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+dynamic+imaging+of+dendritic+spines+using+a+low-affinity+photoconvertible+actin+probe.">Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe.</a> PLoS ONE. 6, (2011).</li></ol>

作者什么都没有透露。

在这篇文章中的工作协议从大鼠原代海马神经元使用SIM卡在体外培养的图像树突棘描述。在原代海马神经元培养方法是通过Kaech和庄家18中描述的原始方法的改编。的主要差别是使用Neurobasal/B27培养基中，从而消除了星形胶质细胞培养物进料器的要求，并且除了在第3天的有丝分裂抑制剂FUDR能促进神经元的存活，同时抑制神经胶质增生的，如所描述由Brewer 等人 19。 <...

一个树突棘是神经元膜的小突起。这种结构的特点是专业，如从单一的突触接收输入并代表两个神经元之间的物理接触面积。功能最成熟的树突棘包括一个球状尖，称为头和颈细，头部连接到树突状轴。然而，刺不是静态的，积极移动，不断改变自己的形态，甚至在成人大脑2。在规定时间内有2周的时间，从胚胎后期或出生后早期的时间得出大鼠原代海马神经元培养开发复杂的树突状乔木与众多的膜突起，从早期的filipodia演变为棘状结构3。在此基础上动态行为等特点，树突棘被认为是提供了一个解剖基板的内存存储和突触传递4,5。 鉴于日Ë关键作用的树突棘的大小和形状都在突触功能，它准确地测量它们的尺寸是很重要的。刺从约200到2,000纳米，长度各不相同，可以通过共焦激光扫描荧光显微镜很容易识别。然而，长度比其他脊椎尺寸通常低于传统的光学系统的分辨率，理论上固定衍射约200纳米的6。这些解决权力是不够的成像更精细的细节，如脊柱的脖子和头的宽度。大量的工作一直致力于解决这个问题，许多相对较新的超高分辨率显微技术提供了实质性的进展。特别是，能够实现超越经典极限分辨率，不通过在一个宽视场，非共聚焦显微镜7-10采用横向结构照明显微镜（SIM）丢弃任何发射光。与非线理组合使用这种技术Ř显微技术，它在理论上是可能通过一个无限因子11，以提高光学显微镜的横向分辨率。然而，在大多数实验的情况下，SIM允许将超过由两个1的因子的分辨率极限。如受激发射损耗（STED）显微镜12和光激活定位显微镜（PALM）12其他超分辨率光学显微技术已被应用到树突棘的影像。定位为基础的方法，如PALM需要非常大的数字原始图像，实现超分辨率的，因此在有限的速度。另一方面，受激发射损耗可以达到高的成像速度，但是在相对 ​​低的光子数和视场小，这可能不是SIM卡13的情况。 在本文中，目的是从大鼠原代海马神经元在体外培养提供了一个工作协议到图像树突棘</EM&gt;使用SIM卡。该协议由两个可区分的阶段：一个初始组成的建立，发展，转染大鼠原代海马神经元培养的免疫组化和专用采样成像的后期阶段。

<table>
	<tbody>
		<tr>
			<td>Fine forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Big forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Big scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope with illumination</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow cell culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-temperature dry-oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator (5% CO2, 37 &#176;C)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon structured illumination microscope setup consisting of:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti research inverted microscope with Perfect Focus System</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon CFI Apo TIRF 100x oil objective lens (N.A. 1.49)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 Coherent Sapphire Lasers (458, 488, 514 and 561 nm exitation wavelength)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SIM Illuminator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Stage Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MCL Nano-Drive piezo controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Intensilight C-HGFIE mercury lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SIM Microscope Enclosure temperature control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Andor EM-CCD Camera iXon DU897</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PC with Microsoft Windows 7 Home Edition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon&#8217;s NiS Elements 6.14 SIM software package</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon type A immersion oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy

树突棘是新兴突起从一个神经元的树突和代表在脑兴奋性输入端的初级突触后指标。技术进步已经确定这些结构在神经元连通性和突触可塑性的关键要素。利用光学显微镜棘形态的定量分析仍然是由于光的折射内在限制相关的技术限制，一个重要的问题。树突棘可以很容易地确定共聚焦激光扫描荧光显微镜。然而，测量在刺的形状和大小的细微变化是困难的，因为比长度其他脊柱的尺寸通常比固定为200nm的光镜的理论分辨率极限传统的光学分辨率小。 几个最近开发的超分辨率技术已被用来对图像蜂窝结构比200纳米，包括树突棘小。 Ŧ淡褐色技术是基于经典的远场操作，因此允许使用现有的样品制备方法及图像以外的试样的表面上。这里描述的是工作协议，超分辨率结构照明显微镜（SIM）适用于原代海马神经元培养树突棘的影像。 SIM卡可能的应用重叠与激光共聚焦显微镜。然而，这两种技术提出不同的适用性。 SIM卡可提供更高的有效横向分辨率，而共焦显微镜中，由于一个物理针孔的使用量，实现了分辨率的改善以除去离焦光的费用。在这个协议中，原代神经元是使用标准协议，转染的DNA的质粒编码的荧光蛋白质和可使用SIM成像的玻璃盖玻片上培养的。这里所描述的整个协议需要大约2周，因为树突棘是体外后16-17天，当成像树突发育是最优的。协议完成后，树突棘可以在3D系列从使用专门的软件，SIM卡图像堆栈的重建。

这里描述的是一种标准化的工作协议，用于从成像使用的SIM 体外大鼠原代海马神经元树突棘。该协议的工作流程，其关键步骤示于图1。总体而言，协议需要大约2周的实验工作中的样品制备的第一阶段，包括文化，发展和大鼠原代海马神经元和免疫组化染分离，并且第二相使用SIM卡的样品成像。大鼠原代海马神经元的文化，当神经元已经开发出复杂的树突状乔木承载无数树突?...

Watch this Scientific Journal Video about Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy at JoVE.com

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

本文介绍了使用结构照明显微镜（SIM卡） 在体外海马神经元的工作协议，以形象的树突棘。使用SIM卡超高分辨率显微镜提供的图像分辨率显著超越在所有三个空间维度的光的衍射极限，允许单个树突棘与改进的细节的影像。

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

imaging-dendritic-spines-rat-primary-hippocampal-neurons-using

Research

JoVE Journal

Neuroscience

15.7K 次观看.  University of Amsterdam. 本文介绍了使用结构照明显微镜（SIM卡） 在体外海马神经元的工作协议，以形象的树突棘。使用SIM卡超高分辨率显微镜提供的图像分辨率显著超越在所有三个空间维度的光的衍射极限，允许单个树突棘与改进的细节的影像。 

视频: Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

科研

神经科学

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

具有突触可塑性树突脊柱的三维建模

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

秀丽隐杆线虫树突棘成像

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

用于登德里脊柱研究的串行块面扫描电子显微镜 （SBEM）

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...