这项研究是由国家科学基金会，神经科学麦克奈特基金，克里斯托弗和Dana里夫基金会和Amerisure慈善基金会的支持。

1。激光烧蚀系统的建设 戴好激光护目镜和使用过程中的初始对准良好的激光安全规范 。 切勿直视激光通过镜片时。 <ol><li>排列图所示的实验电路板的组件。 1（见例2）。螺栓向下的激光（如果需要立管板），潜望镜后，和高架轨道支持。 </li><li>您的显微镜的位置，以配合铁路轴。从显微镜聚光镜传输光束对齐。对齐轨道高度（使用A级）和一个可调节光圈或一个镜头贴铁路的位置。光圈或镜头必须在冷凝器梁铁路两端对齐。实验室插孔，可以帮助调整轨道高度。 </li><li>将格兰 - 汤普森偏光镜和半波片的激光束。您不需要对激光做到这一点对齐。乙EAM只需要通过双方没有触及其边缘。旋转偏光器提供了方便的方向和梁转储的位置（图1）。调整激光功率，您将旋转半波片。实验电路板的安全组件。 </li><li>打开激光，脉冲频率设置为100，连续模式，并减少到最低限度使用半波片的电源。使用后的说明或镜头纸以可视化的激光束。 </li><li>调整和修复运动学安装序列的激光镜，把梁高架轨道上安装镜子。大致镜子的中心应对准激光束。镜子应面向大约45度的激光束的轴（图1）。 </li><li>粗糙调整潜望镜，顶部和铁路接轨的激光束从显微镜透射光束中心结束运动学安装镜子。无论是激光束和T他从显微镜光束传输，可以同时在光束路径中插入文件的可视化。确保任何ND滤镜和光圈在中间模块出或完全开放。 </li><li>精细对准激光束传输的光束中心与顶部潜望镜和铁路镜子的精细调整。按住纸张靠近铁路镜对准激光束顶端的潜望镜镜传输光束的中心。保持显微镜港口附近的纸张，并配合铁路镜调整（图2）激光束传输的光束中心。 </li><li>定位后在光束路径之间的对齐聚光镜和开放的客观炮塔注意。您应该看到，中心附近较小的激光束传输的光束。上使用的轨道镜中心的激光束进行精细调整。修正与夹具的地方显微镜。 </li></ol>步骤1.9-1.12是可选的，但它是简单而有用的，以评估之前加入光束扩大镜头对准和激光烧蚀。 <ol start="9"><li>关闭激光和从事机械安全快门。旋转光束路径的分色出50％。 ，即使没有直接的激光灯将传递到目镜，反射光可以相当激烈。安装优良的路线与60X石油目标图像的目标幻灯片。重点放在幻灯片上的划痕。放在中间模块的ND4和ND8过滤。 </li><li>形象与你CLSM的目标幻灯片，旋转盘，或CCD相机系统。 从这一点来说，你不应该通过目镜而烧蚀激光是激光路径旋转到50％分色。打开激光消融和模式设置为触发，频率为100，和脉冲数为10。确保电源仍然设置为最小的半波片，然后打开机械安全快门。您可以simultaneou狡猾的形象，并使用激光消融。 </li><li>虽然成像的目标幻灯片，触发激光消融。检查消融点在直径1-10 UM的目标幻灯片。直到看到消融点顺序删除ND滤镜。与铁路的镜子，调整消融现场图像的中心。您将要添加一个ND滤镜有房精细增加半波片的激光功率&lt;1微米消融当场。在0.2微米/像素或更低，微调消融现场图像的中心位置（256,256 512x512的图像）的图像。 </li><li>检查的重点和激光束轴线深度。焦点上下1-2微米和检查消融的地位和消融光斑大小。如果激光束的轴向对齐消融点不会移动。无条件的激光束将分布在几个微米功率（约3-5微米，从顶部底部的重点）。 </li></ol> 2。新增镜片扩大激光束来填补目标回光圈和调整收敛控制的重点<ol><li>该系统采用双伽利略扩束扩大的激光束，填补了客观的回光圈（10毫米）。山运营商的4个镜头，将它们附加到铁路（L1f1 + L2f2和L3f1&#39;+ L4f2&#39;）。调整位置，使所有的镜头都在同一光轴和完全正交的激光束（图2）。 </li><li>打开激光和调整，以最小的功率和连续模式。粗略调整，通过每一个镜头使用的纸张看到激光束束对准显微镜聚光光束传输。关闭快门的激光。 </li><li>从一个侧面显微镜取出夹子和幻灯片在显微镜的激光束路径。打开激光和消除安全快门。 </li><li>罚款的镜头和激光束的对齐方式可以扩大激光束通过查看附近的墙壁上。梁应扩大和合同对称勒NSES是感动。调整的最后两个运动学安装镜子对准梁（图3）。 </li><li>对齐和扩束配置文件应该是圆形的，亮度均匀（图3）。与华盛顿邮报"的大小和均匀的光束可以被视为在估计目标回光圈位置的注意。 </li><li>梁应调整到无限远光学系统的收敛为零。收敛可估计注意到如何光束大小的变化，移动后的注意镜片更远。关闭激光和从事机械快门。 </li><li>在显微镜幻灯片回的激光束，使用固定夹具定义正确的对齐路径。显微镜自由更换夹具，但不要拧紧。 </li><li>打开激光和消除安全快门。客观炮塔旋转到打开位置。放置后，它在舞台上看到。您应该会看到激光束透射光b为中心的扩大和统一从冷凝器EAM（图4）。您可能需要中心的IT松动显微镜夹，小心轻推显微镜。 </li><li>搞的安全快门，并设置激光触发模式。旋转60X目标到位，形象的目标幻灯片上的表面划伤。 </li><li>删除的安全快门，触发激光消融和激光功率调整为最低消融点。消融当场应该是圆形的，并在图像的中心5-10微米。 </li><li>中心与消融当场罚款运动学安装的铁路镜调整。您将需要反复调整导轨安装和顶部潜望镜镜都中心的消融点，并保持均匀的光束轮廓。 </li><li>评估将焦点移动系统上下在1微米的步骤和触发激光消融，激光的Z焦点。扩大，对齐和收敛的调整梁应最大限度地消融在图像的焦点，弱或没有在所有1微米的ABOVE或以下重点（图5）。 </li><li>调整移动镜头伽利略望远镜（图）L3 ž扩束的焦点。 </li></ol> 3。激光干切断和轴突再生的时间推移显微镜<ol><li>微波10％琼脂糖（RPI的分子生物学级平等就业机会0.1 A20090）在平衡盐，直到完全融化。设置一个热点板块在使用过程中保持它融化，用于安装。 </li><li>一滴熔化的琼脂糖置于载玻片上，并与另一载玻片成约200微米厚（间隔使用的时间磁带上相邻的幻灯片单层）垫夷为平地。 </li><li>标记第一个"Sharpie"是用来切出一个穿制服的直径为13mm的圆形垫。 </li><li>麻醉（1ul蝇蕈醇20MM）和微球（克里斯芳日元，个人通讯）（1ul 2.65％聚苯乙烯水在0.1微米）被添加到3-5蠕虫面向垫的中心，所以他们就躺在他们的左右两侧。适用于玻璃盖玻片，然后VaselINE是用来密封盖玻片，防止样品蒸发（图6）。 </li><li>烧蚀激光对准十字线，如上所述，在每届会议开始。从事激光安全快门。 </li><li>拆下激光对准目标与一个合适的目标神经元荧光标记和图像安装成虫。图片轴突类似的放大倍率（0.2微米/像素或更高的放大倍率）和下十字线（图7）的位置。 </li><li>打开激光安全快门触发而成像的消融激光。评估后触发激光的轴突和慢慢增加激光功率，直到被切断的轴突。您将需要约10倍以上的激光功率削减比轴突形成一个对准目标幻灯片（约1uJ / ns脉冲）的消融现场。我们通常会削减100个脉冲，在2.5 kHz和功率设置约0.27mW（标本相干FieldMaxII在2500赫兹功率计和激光连续平均功率测量）。消融激光功率设置将在以后的实验中（图7）切割轴突一致。 </li><li>一个成功切断的轴突会显示没有休息约0.5-1微米的亮度在两个切端（图7）的损失。大量的亮度或相当大的差距（2-10微米）的损失往往意味着超出了由空化泡的轴突广泛的破坏。近端和远端的轴突会分开，形成回缩灯泡在接下来的30分钟（图8和电影）。 </li><li>您的显微镜阶段平衡时间的推移录制开始前30分钟你安装的蠕虫病毒。这将最大限度地减少由于温度差异和琼脂糖凝胶收缩的漂移。 </li><li>使用与您的系统相关的图像处理软件）定时成像参数。我们通常形象10-20 Ž步骤0.1-0.2微米/像素（1um/step）使用增益，针孔，扫描速度和成像激光功率设置，最大限度地减少曝光，同时提供所需的空间分辨率。采样间隔是典型的LY 1-5分钟，超过15小时，取决于所需的时间分辨率（图8和电影）。 </li><li>时间推移图像数据转换成使用NIS元素或ImageJ的电影。 </li></ol> 4。代表性的成果: 激光干切断，使用这个系统是一个可靠的和日常。如图7所示的结果，是典型的。定时成像轴突再生是非常强大，使用该协议。我们经常剪切和图像5轴突在5个不同的蠕虫使用机动阶段在一个幻灯片。唯一的限制是需要收集每个轴突，例如图像，如果需要20秒收集轴突堆栈，然后在大多数情况下，您可以品尝9轴突（9栈）如果你是每180秒采样时间。在图8和图9所示的例子是一个很好的代表的结果。约10％的实验，给这个质量的结果。其余实验提供良好的数据再生的表型，但美观unappealING因为小抖动的蠕虫病毒，一般固定5-8小时后开始运动。 <img alt="图1" src="/files/ftp_upload/3331/3331fig1.jpg" /> 图1激光烧蚀系统。 532 nm的Nd:YAG激光器安装在立管板，将其带到其他组件的高度和螺栓的面包板。 （光隔离器是可选的，可能不需要）。格兰 - 汤普森偏光镜和半波片是用于精确控制激光功率。他们精心校准的旋转支架。小心设置束转储。角镜引导激光束，以较低的潜望镜镜。上潜望镜镜的光束定向铁路镜。这条铁路是安装在两个杆支持的平台。双伽利略镜头连接到滑轨支架。注意补充的中间模块专用激光消融。该系统可以去除光隔离更加紧凑Tor和角落的镜子，和重新定位，格兰 - 汤普森激光偏光器和半波片的实验电路板的背面边缘。 <img alt="图2" src="/files/ftp_upload/3331/3331fig2.jpg" /> 图2双伽利略调节镜头是用来扩大300 UM TEMoo激光束零衔接统一的10毫米大小。实验室插孔定位下的铁路平台之一。这有助于调整轨道高度在对齐步骤。粗略地调整，由安装在L1镜头铁路和使用冷凝器束作为定位导向的轨道高度。调整轨道，使冷凝器束的镜头在铁路两端的中心对齐。这将确保铁路平行光轴显微镜。您可能需要先调整显微镜相匹配的铁路光轴。使用夹子修复显微镜的位置（见图1）。删除第发送轨安装镜头。现在排列在铁路两端的中心冷凝器束激光束。在光束路径的文件，是一种方便的方法，同时看到两束。使用顶部的潜望镜镜对准激光束的聚光光束的轨道式镜。使用导轨安装镜对准铁路的尽头（最接近显微镜）冷凝器束激光束。现在安装所有四个镜头，图中所示的轨道。铁路的长度，焦距的镜头，和镜头的安排会有所不同取决于你的显微镜和物理设立。关键参数的激光束的直径和所选择的目标回光圈的大小。无限远光学系统将集中一个完全平行的激光束（零趋同）。 L1和L2之间的距离应在镜片的总和，焦距，F1 + F2，L3和L4之间的距离应相应F1"+ F2"。日之间的距离E双伽利略对并不重要。 L3的位置可微调控制射束会聚&lt;1毫米调整将需要调整激光图像的焦点的焦点。请务必从中间模块可能在光束路径（或知道他们做什么，并相应地调整你的系统）中删除所有的镜头，过滤器，孔径等。 <img alt="图3" src="/files/ftp_upload/3331/3331fig3.jpg" /> 图3通过双伽利略镜头对准激光束。删除显微镜夹，以便从一个侧面显微镜显微镜可移动的光束路径，但其余的夹子将允许在显微镜精确的重新定位。认为激光安全。确保格兰 - 汤普森偏光镜激光功率调整到最低限度。打开激光，查看墙壁上的激光束模式。您可能会发现贴在墙上的一纸，有助于查看激光现场。系统地调整镜头位置，以获得为他们做什么束的大小和外观的感觉。您应该看到的东西，看起来像面板，激烈的地方是偏心调光器配置文件位于。使用轨道式镜面板B.现在使用顶部的潜望镜所示调整激烈的现货中心安装镜子给一个统一的外围轮廓。如果一切都是正确对齐的，你现在应该找到那动人的镜头，使光束对称扩张和收缩。将镜头回到起始位置如图2所示。现在拦截在距离目标回光圈的光束。它应该是圆形的，至少大到足以填补你的背部光圈，一个统一的亮度。这是确定的，如果是较大的，只要你有足够的激光功率为您的消融。评价比较客观回光圈和墙壁的距离光束直径的衔接。光束直径应零收敛束相同。比例尺为10mm。 <img alt="图4" src="/files/ftp_upload/3331/3331fig4.jpg" /> 图4扩展的激光束对准冷凝器束。快门的激光和显微镜移动到对齐停止使用夹具的激光束的路径。打开，对齐和重点使用的目镜一个客观的镜头。 胶带纸，以防止任何人观看反映强烈的激光灯的聚光光束。客观炮塔旋转到打开位置。在舞台上放置一张纸堵塞冷凝器束。打开连续模式的激光和删除机械安全快门。您应该看到一些激光光显微镜虽然。松开显微镜夹，轻轻微调，以配合冷凝器束激光束显微镜。激光束应该是圆形的和均匀的亮度。有时是有用的佐剂ST的轨道式镜头。你应该能扩张和收缩的激光束均匀如果对齐是正确的。您可以中心的签约最低中心的聚光光束激光束与导轨安装镜像，然后扩大到所需的大小。一旦你调整与轨道镜承包梁的位置，您将需要调整的顶部安装的潜望镜镜，保持扩束的亮度均匀。如果你不能得到一个圆中心的激光束，那么你将需要通过早期的对齐步骤。 <img alt="图5" src="/files/ftp_upload/3331/3331fig5.jpg" /> 图5中心的消融点，并调整为最佳聚焦的激光束收敛。关闭快门的激光。删除的文件和挂载镜像的目标幻灯片（你也可以使用在盖玻片作为目标2记号笔墨水）。与60的镜像表面上的图像划痕X 1.4na石油镜头）现在的形象与焦或CCD表面。 别看通过的镜片，而激光。打开和unshutter激光。设置激光触发模式，功耗最低，和100 Hz。约10个脉冲触发。您应该看到一个中心的观点，在10微米直径1-5微米的消融当场。如果您没有看到消融点，你可以增加激光功率在5-10％的步骤。一旦你确定消融点，中心视野。如果观看512x512的图像，把256，256坐标十字线。如果调整激光光斑中心，然后它会不会改变与缩放的位置。使用导轨安装镜子的中心十字线移动消融当场。重新评估删除在上面的图4描述的客观梁的激光束的均匀性。调整与前潜望镜装镜的光束均匀。重复评价消融当场表态。这是一个迭代process，应反复进行，直到消融点为中心，扩束均匀。如果做得正确消融当场将圆在图中的焦点消融当场看到。接下来，评估在Z轴方向的激光束的焦点。触发激光生产中心约1微米的消融当场。现在移动约5微米的幻灯片，横向和重点目标幻灯片表面以下1 UM。触发，使用相同的激光设置作为您首次消融当场使用激光。聚焦后1微米以上的目标幻灯片表面的重复。如果激光束聚焦到图像的焦点，那么你应该看到这个数字表明，类似的模式。最大的消融当场应该对应于图像的焦点和消融点的上方和下方的重点应该得到较小的对称。调整镜头L3调整焦平面。从显微镜移动镜头L3的距离将梁收敛和消融点靠拢的目标。小&lt;1毫米调整将需要近的焦点。您现在准备削减轴突。比例尺为1微米。 <img alt="图6" src="/files/ftp_upload/3331/3331fig6.jpg" /> 图6安装蠕虫激光干切断和时间推移成像。琼脂糖10％的生理盐水中加热至完全融化。一小滴被放置在玻璃显微镜幻灯片，然后用另一张幻灯片夷为平地，形成一个平垫。垫的厚度是由磁带上的两个相邻的幻灯片。垫是可以设置为1分钟左右，然后是分开的幻灯片。一个"Sharpie"的顶部是用来作为一个俗套，形成一个统一的大小约13毫米垫。 1 UL 10MM蝇蕈醇微球和1个UL垫中心（颜芳的个人通讯）。蠕虫被添加到2 UL下降，面向前把玻璃上的盖玻片。凡士林，然后从注射器（20-25 GA。针）盖玻片密封的边缘应用。 <pCLASS ="jove_content"&gt; <img alt="图7" src="/files/ftp_upload/3331/3331fig7.jpg" /> 图7激光线虫干切断的典型结果。你可以看到完整的轴突在不久之前和之后激光干切断。差距是正常约0.5 - 1um的。 B显示不破坏附近的轴突，相距约1微米的激光之一轴突干切断。激光设置为10％左右的功率（0.3毫瓦平均在2500赫兹），100个脉冲。比例尺为5微米。 <img alt="图8" src="/files/ftp_upload/3331/3331fig8.jpg" /> 图8。典型的野生型L4再生。这是一个时间序列，显示消融和时间推移轴突再生的录音。激光干切断后不久，在0分钟，你可以看到回缩球。 138分钟回缩球拉回最远的程度，现在开始改建。零星的细胞膜突起（mircospikes或丝状伪足）可以看出，沿着轴突的轴（箭头138和324）。 "正确的残端形成良好的紧凑型生长锥延伸，在360分钟。在414分钟延长两个树桩的生长锥。最初的胚胎样生长锥成为营养不良，因为他们延长朝着背神经索（414，519和597分钟）。营养不良性生长锥摊位背神经索（960分钟）。箭头指出近端残端和生长锥。箭头指出沿近端轴突的轴膜突起。比例尺20um。 电影1。野生型轴突再生的影片。这部电影随之而来，如图8所示的时间点。蠕虫安装中所述的协议和轴突成像约10小时，每三分钟。 ž栈（20 × 1微米）在每个时间点采取了最大的投影算法（NIS元素）合并成单一的图像<a href="http://www.jove.com/files/ftp_upload/3331/3331movie1.mov">。点击这里观看电影</a> 。

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没有利益冲突的声明。

不同的激光系统3,5-11有几个不错的激光显微讨论。飞秒红外激光器是12和方便，如果成像设备的亚细胞激光消融的"金标准"，但他们往往是个人用户的成本太高。如果您需要飞秒红外激光成像你的样品，因为成像深度，然后你可能会需要一个激光干切断。在30-50微米的表面与目标的轴突透明组织可能在20 UJ /脉冲通过有针对性的高NA浸没透镜的范围的蓝色和绿色脉冲激光器是可行的。附带损害与纳米和pico第二激光器与飞秒激光相比将会有更多的，尤其是作为目标轴突增加深度C。线虫是透明的，所有的轴突在20-30微米的表面。我们经常切断电机轴突在5微米的表面。我们也轻松晋级AX组件内的神经环，约20微米的表面角质层。我们发现切割轴突通过成虫的直径约30-50微米的限制。它是可能的，这里描述的激光消融系统的工作目标轴突的透明度和深度的标准的许多适合不同制剂。尽管如此，它是有点出人意料，由于飞秒红外激光器，纳米和皮秒355纳米和532纳米激光器的理论优势执行使激光在三干切断线虫 6,13。我们看到，在纳秒440纳米，纳秒级的532纳米，和飞秒红外激光器激光干切断轴突再生无差异。 固态355 nm紫外激光器相同的成本大约为532 nm的二极管泵浦被动调Q固体激光器，但需要更高的权力或光学系统，有效地传递这些波长较短的。大多数光学设计以及执行可见400 -700 nm的光。 6，如降低血浆门槛，光斑尺寸较小，长传分色，有效地将直接激光消融的100％的目标，并允许同时成像，无样品信号损失的GFP 355纳米激光器将提供一定的优势。蓝440纳米激光器将保留可见光激光器（与标准的光学和安全性能好）工作的优势。不幸的是，民进党Q开关固态440 nm激光的成本是355纳米或532纳米的一个类似的权力，在这个时候的激光成本的3倍。如果我们在设计一个新的系统的 C 线虫 ，目标深度是最小的，我们会选择在1的紫外线的透明镜片（成本50-70％的透光率在355纳米一个40X 1.3na石油镜头3000元左右）和355 nm激光生产5-10 UJ /脉冲-20千赫。 Axon的消融被认为是由于等离子体形成。过短的脉冲会产生等离子体的阈值，低功耗和血浆量瓦特<html>生病是更小的6。我们的目标是通过调整脉冲能量最低的功耗最小和寿命最短的等离子生产。较大的长寿命等离子体将产生空化气泡周围细胞的损害。我们普遍发现，使用频率最高的（即2.5kHz）约50-100脉冲的消融提供了最好的结果。这表明，损害可逐渐整合一系列的脉冲，以更好地控制损害程度。这里描述的激光消融系统是非常宽容的，如果光束准确对齐和扩大。我们开始削减10％的激光功率（0.3毫瓦平均在2500 Hz的测量和估计1 UJ /脉冲）在成虫轴突，可以削减14％的电力有限，通过局部损坏。你可以观察到从15-39％的激光功率的损害较大的地区，但只有40％以上，功率（约1毫瓦平均在2500赫兹测量）蠕虫爆炸，由于大型空化气泡和蠕虫的角质层损害。 e_content"Steinmeyer等。2010年2纸张是一个极好的资源，为建设一个激光消融系统。莱昂艾利也有很好的实际描述的一种廉价的氮气337 nm激光的激光烧蚀系统，他提供了一些重大的现实意见（elegans.swmed.edu / / Worm_labs /艾利），设计自己的系统时，你应该先谈谈您的显微镜代表，以确定如何定位消融激光显微镜目标。您的显微镜应安装隔振表（纽波特，TMI，托尔）。面包板的顶部是最优的，但仍然可以使用磁定位实心钢顶。一个堂堂正正的范围的中间一个专门的模块是好的，因为所有的光学元件，可以留在原地，但是，它可能更便宜和更方便地使用相机端口（倒）或"合成"EPI -荧光端口，你需要知道后面的光圈和透光率的大小（在A<html> blation激光波长）的物​​镜，你打算使用。一旦你决定用激光（例如，Crylas，天河城，水晶，或CRC），您应该联系选择正确的光学元件，硬件和安全设备的帮助雷神或新港。我们决定在双伽利略扩束，以节省空间，但一个简单的开普勒扩展选项（f2/f1 =膨胀系数）。自定义的扩束，将是最便宜的，但新港，雷神，和几个激光制造商提供优良的品质商业扩束。一些激光制造商还提供手动和电子控制的衰减器，可能是成本效益和节省空间，但不能作为多功能格兰 - 汤普森偏振片及半波片。您还需要来决定如何控制激光。您可以设计一个基于LabVIEW的控制器，使用商业TTL发生器，或由激光公司提供的软件。讨论与特定的激光制造商的选项。 帐篷"&gt;我们已经提供了我们有用于作为一般指南只的硬件列表。系统描述在这里成本约一万五千元到我们现有的成像系统中。您的本地物理部门会经常有有人愿意来提供的实用介绍到安全工作与自由束激光。 C 的固定时间的推移成像的替代方法线虫最近已描述。14 故障排除最困难和最关键的一步，是中心的扩束显微镜的光轴对齐。一旦你通过伽利略镜头，你应该努力在5显微镜的光轴UM它对齐，然后使用该中心的最终对齐转向镜中心和扩大梁。转向后视镜的过度使用显微镜对准光束通过光束将它移到轴扩展。 使用格外小心 ，你可以衰减适当的ND过滤器的激光束和激光光束剖面上反射目标（前表面反射镜）直接查看。你可以轻轻地推动了正是中心60X目标领域的光束显微镜（如果它不能在向上/向下范围，你需要调整轨道高度为中心）。光束轮廓可用于精确调整显微镜的光学轴，一个中心束循环和对称的"信号弹"。非对称耀斑是显微镜轴不完全一致（或铁路是不是水平）。最后，你应该调整L3和看到光束均匀，对称的扩张和收缩。精确聚焦显微镜的目标表面上，然后调整L3集中的激光束，以最小的位置。您现在应该发现，是通过激光共聚焦显微镜或CCD系统成像和消融点在激光束在5个中心UM1微米的Ž焦点。 如果你发现你是"吹"蠕虫或始终如一地产生大量的空化气泡削减轴突您的问题是最有可能的罚款消融激光对齐。确保最低（&lt;500nm处）消融点定位到Z的重点（调整L3的镜头衔接）和XY受托现货（与去年运动学安装镜子调整）。 瞄准轴突更接近表面，将需要较少的激光功率。同样，小动物（L1和L2）将需要少干切断激光功率相比，针对成年人同样的轴突。 如果您的野生型轴突没有再生或轴突漂白剂，你应该尽量减少成像激光功率或减少采样率。如果您的蠕虫死亡或病弱的尝试，减少1％％琼脂糖。当使用高每确保你是不是安装后，蠕虫移动盖玻片，因为这往往会导致他们死百分比琼脂糖微球。 如果您的蠕虫爆裂或在一个时间推移会死，这是由于:1。百分比琼脂糖过高。 2。烧蚀激光伤害到角质层。 3。安装后的盖玻片运动。如果角质层受损有过错的，它只会影响安装已与激光消融针对性的蠕虫。 如果您的蠕虫移动太多的尝试，在一个时间推移会议在0.5％的步增加的百分比琼脂糖。健康蠕虫健康轴突总是"积极"和显示的荧光一致的水平。如果你看到一个荧光或活动突然减少蠕虫是垂死或死亡。多个串珠或零散的轴突是一个垂死或死亡蠕虫的明确迹象。 如果你有麻烦您的轴突在整个时间的推移会保持对焦，可能有几个不同的问题。首先由成像检查阶段漂移载玻片上惰性的样品超过10小时RS。几微米的漂移可能是由于热不稳定性，但大于5微米，可能是由于机械问题与您的显微镜。日益严重的问题是比较常见的。让您安装蠕虫平衡与您的显微镜阶段开始前30分钟时间的推移。检查您的凡士林密封的发展并不在时间的推移会话过程中的泄漏。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> 535纳米激光</td><td> Crylas </td><td> FDSS532Q3 </td><td> （天河城，水晶，和CRC还提供媲美激光） </td></tr><tr><td>所有的光学和硬件</td><td></td><td></td><td> （托尔提供媲美光学和硬件） </td></tr><tr><td> SUPREMA光学山，直径1.0 - </td><td>新港</td><td> SS100 - F2KN </td><td> 2 </td></tr><tr><td>直径为1"后，1"的高度</td><td>新港</td><td> PS - 1 </td><td> 1 </td></tr><tr><td>直径为1"后叉</td><td>新港</td><td> PS - F </td><td> 1 </td></tr><tr><td>消色差零阶沃维勒板，半波迟缓，400 - 700nm的</td><td>新港</td><td> 10RP52 - 1 </td><td> 1 </td></tr><tr><td>旋转舞台，1"Aperature </td><td>新港</td><td> RSP - 1T </td><td> 1 </td></tr><tr><td>直径为1"后，1"的高度</td><td>新港</td><td> PS - 1 </td><td> 1 </td></tr><tr><td>直径为1"后叉</td><td>新港</td><td> PS - F </td><td> 1 </td></tr><tr><td>方解石格兰激光偏光，430 - 700nm的</td><td>新港</td><td> 10GL08AR.14 </td><td> 1 </td></tr><tr><td>偏光旋转安装</td><td>新港</td><td> RM25A </td><td> 1 </td></tr><tr><td> ¼"间隔1"直径后</td><td>新港</td><td> PS - 0.25 </td><td> 1 </td></tr><tr><td> ½"的高度，直径为1"后</td><td>新港</td><td> PS - 0.5 </td><td> 1 </td></tr><tr><td>叉，1"直径后</td><td>新港</td><td> PS - F </td><td> 1 </td></tr><tr><td>梁转储</td><td>新港</td><td> PL15 </td><td> 1 </td></tr><tr><td>显微镜物镜镜头安装</td><td>新港</td><td> LH - OBJ1 </td><td> 1 </td></tr><tr><td>直径为1"后，1"的高度</td><td>新港</td><td> PS - 1 </td><td> 1 </td></tr><tr><td>直径为1"后叉</td><td>新港</td><td> PS - F </td><td> 1 </td></tr><tr><td>高能量的Nd:YAG激光反射镜，直径25.4毫米，45 °，532纳米</td><td>新港</td><td> 10Q20HE.2 </td><td> 4 </td></tr><tr><td> SUPREMA光学式，1.0英寸直径，清除安装孔</td><td>新港</td><td> SN100C的- F </td><td> 2 </td></tr><tr><td>高精度旋钮调节螺丝，12.7毫米旅游，100TPI </td><td>新港</td><td> AJS100 - 0.5K </td><td> 4 </td></tr><tr><td>螺纹接头，¼ -20男，女8-32 </td><td>新港</td><td> SS - 1 - B </td><td> 2 </td></tr><tr><td> 1.5英寸直径的棒杆钳</td><td>新港</td><td> 340 RC </td><td> 2 </td></tr><tr><td>棒，身高14英寸（35.5厘米） </td><td>新港</td><td> 40 </TD&gt; <td> 1 </td></tr><tr><td>铁路承运人为X26，方40毫米长度</td><td>新港</td><td> CN26 - 40 </td><td> 4 </td></tr><tr><td>钢轨，384毫米（15"）长度</td><td>新港</td><td> X26 - 384 </td><td> 1 </td></tr><tr><td> 1.5英寸直径的棒杆纲要</td><td>新港</td><td> 300 - P </td><td> 2 </td></tr><tr><td>棒，身高14英寸（35.5厘米） </td><td>新港</td><td> 40 </td><td> 2 </td></tr><tr><td>平凹透镜，12.7毫米直径- 25MM，430 - 700nm的英语</td><td>新港</td><td> KPC025AR.14 </td><td> 2 </td></tr><tr><td>平凸透镜，25.4毫米直径，50.2毫米英语，430 - 700nm的</td><td>新港</td><td> KPX082AR.14 </td><td> 1 </td></tr><tr><td>平凸透镜，25.4毫米直径，62.9毫米英语，430 - 700nm的</td><td>新港</td><td> KPX085AR.14 </td><td> 1 </td></tr><tr><td>固定镜头卡口，直径为0.5"，1.0"轴高度</td><td>新港</td><td> LH - 0.5 </td><td> 2 </td></tr><tr><td>固定镜头卡口，1.0"直径1.0"轴高度</td><td>新港</td><td> LH - 1 </td><td> 2 </td></tr><tr><td>微0.1um </td><td> Polysciences </td><td> 00876 </td><td></td></tr><tr><td>琼脂糖</td><td>零售物价指数</td><td> A20090 </td><td>平等就业机会的事宜</td></tr><tr><td>蝇蕈醇</td><td>西格玛</td><td> M1523 </td><td></td></tr></tbody></table>

constructing a low-budget laser axotomy system to study axon regeneration in c. elegans

激光干切断时间推移显微镜是一个敏感的检测轴突再生的表型在 C 线虫1。这个实验的主要困难是感知的成本（$ 25 - 100K）和实施激光烧蚀系统2,3所需的技术专长。然而，适度的成本（&lt;$ 10K）固态脉冲激光器可以提供强大的性能在透明的筹备工作目标轴突是"接近"组织表面的激光消融。在一天内可以完成系统的建设和对齐。光提供从集中冷凝器消融激光光路提供了一个方便的定位导向。中间模块可与全部拆除光学专用激光消融和保证，没有光学元件需要激光烧蚀会议期间提出的。在中间模块分色允许同时成像和激光烧蚀。围绕激光束吨Ø传出束聚焦显微镜聚光镜引导系统的初始对准。各种镜头的使用条件和扩大的激光束，以填补选择物镜后孔径。最终校准和测试执行与前表面镜面玻璃幻灯片的目标。激光功率调整到最小尺寸消融现货（&lt;1微米）。消融点为中心的运动学安装的最后镜微调跨头发固定在成像窗口。激光干切断电源，将约10倍高于最低目标幻灯片（这可能会随您使用的目标）消融当场需要。蠕虫可以通过安装在琼脂糖垫（或在微流体商会4）固定激光干切断和时间推移成像。 10％的琼脂糖琼脂糖垫很容易在微波炉中融化的平衡盐。一滴熔化的琼脂糖置于载玻片上，并与另一夷为平地玻璃垫成幻灯片，约200微米厚（单层的时间磁带上相邻的幻灯片，是用来作为一个间隔）。第一个"Sharpie"是用来切出一个穿制服的直径为13mm的圆形垫。麻醉（1ul蝇蕈醇20MM）和微（克里斯方颜个人通讯）（1ul 2.65％，聚苯乙烯水在0.1微米）添加3-5蠕虫导向，所以他们就躺在他们的左右两侧垫的中心。适用于玻璃盖玻片，然后凡士林是用来密封盖玻片，防止样品蒸发。

Watch this Scientific Journal Video about Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans at JoVE.com

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

激光干切断时间推移成像是一个敏感的方法检测基因突变的影响<em&gt; C。线虫</em&gt;轴突再生。一支素质高，但价格低廉，激光消融系统可以很容易地添加到最显微镜。超过15小时的时间的推移成像需要小心固定的蠕虫病毒。

构建一个低预算的激光干切断系统研究轴突再生 C。线虫</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

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Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

15.4K 次观看.  University of Utah. 激光干切断时间推移成像是一个敏感的方法检测基因突变的影响 C。线虫轴突再生。一支素质高，但价格低廉，激光消融系统可以很容易地添加到最显微镜。超过15小时的时间的推移成像需要小心固定的蠕虫病毒。

视频: Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

在胎儿肺细胞的机械传导的实验系统研究

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.1 We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.
Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.2
C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.3 The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.4 We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.

Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.

使用激光衍射自由游泳微生物的定量步态研究

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

在激光诱导轴突病变张力释放测量评估轴突在Piconewton和毫秒级的分辨率对基材的附着力

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中脉冲激光烧蚀制备金属纳米粒子:研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...