我们感谢S.托马斯谁发起了这个项目，J.赫尔曼和U. Burgbacher在FIM安装施工的帮助。这个工作是由东风集团（SFB 629 B6）。

注：在这里，使用FIM被提交幼虫运动的筛查自由移动条件下热刺激的影响，方便高通量分析。其他应用，如嗅觉刺激依赖运动或滚动或其他行为的高分辨率成像可能需要微妙的变化对协议，该协议可应要求提供的分析。 1.建立实验<ol style="margin-left: 40px;"><li>使用共（需要1小时的时间）每型约100幼虫获得统计学意义的价值观。注意：如果几个基因型需要比较并记录在不同的日子，记录每天每基因型幼虫的数量相同。 </li><li>对于大多数应用，在10赫兹的参数分析记录。对于高分辨率成像应用中，如果需要改变成像速度。 </li><li>第三龄幼虫，大约产卵后的120小时进行试验的跟踪。让河畔Ë的文化产生足够的幼虫在实验期间（见4.3）。 </li><li>对于非刺激应用，制备含半透明的琼脂表面爬行（见第2节）。含有幼虫的刺激范围为热梯度的应用，环绕的视场与厌恶盐琼脂屏障（参见第3节）。 注：其他应用可能需要不同的表面。 </li><li>请务必使用一个带恒定的环境条件（温度，光照，气流，空气湿度等 ）。 </li></ol> 2.爬行表面处理（半透明琼脂） 注：在FIM设置的琼脂表面加入提供了潮湿的爬行表面。此外，它也提高了照明属性。 <ol style="margin-left: 40px;"><li>煮沸0.8％食品级琼脂在去离子纯水。对于筛选的应用做好准备400毫升。 </li><li>在一个单独的33厘米X33厘米交流倾琼脂在50℃下（手 - 热）rylic-玻璃板。琼脂的表面张力，因此，温度将限定琼脂板的厚度。在50℃，2毫米厚的琼脂板被获得。煮沸后不要搅拌，倒入不断以避免气泡。准备新鲜的琼脂板为约4小时每拍摄期间。 </li><li>填写标准的6毫米培养皿中，其余0.8％的琼脂溶液进行排序，清洁和幼虫，观察者实验（每基因型1盘）之前。 </li><li>如果厌恶琼脂障碍是需要的应用程序，进行第3节。 </li><li>修剪过的琼脂板的周界的约2cm，以获得一个平面方形表面进行记录。删除多余的琼脂。 注：大小是依赖于应用。 </li><li>轻轻推滑行琼脂在有机玻璃板的边缘在冷却后直接传输琼脂板的FIM设置。 </li></ol> 3.可选：添加厌恶琼脂屏障，克劳林志玲表面（盐琼脂） <ol style="margin-left: 40px;"><li>煮沸的2.5％的食品级琼脂用3M NaCl的去离子超纯水。用于筛选在热梯度制备200毫升 注：体积取决于应用程序。 </li><li>切2厘米宽的缺口，先前倒周围的景色（22×22厘米）领域的爬行表面。 注意：不同的屏障的形式和视场可根据应用需要。 </li><li>填写盐琼脂比跟踪表面高0.1-0.3厘米的槽口。 </li></ol> 4.飞处理<ol style="margin-left: 40px;"><li>后方苍蝇在25℃培养箱中以12小时光照/黑暗周期，在65％的空气湿度调节到实验的时间标准飞食品。 </li><li>对于十字架，麻醉30处女雌性果蝇和雄性8与CO 2和交叉他们130 ml培养瓶35毫升食物。 注意：一个130毫升文化小瓶35毫升食物将产生约20-30升4小时的成像范围内连吃三龄幼虫。成像和跟踪工作全部龄幼虫阶段。 </li><li>滴一点水文化小瓶带动后期三龄幼虫运动和收集利用小画笔小瓶墙壁上最大的幼虫。 </li><li> 2-5分钟录制前，一个视频含0.8％食品级琼脂在去离子超纯水使其习惯和清理他们一个培养皿转移幼虫。 </li></ol> 5.调整FIM成像设置的录音（非条件刺激） <ol style="margin-left: 40px;"><li>如果需要调节摄像头的镜头焦距和光圈。设置曝光时间对于各摄像机。 注意：不需要实验过程中要改变这些设置。 </li><li>调节照明强度由成像1幼虫，以获得良好的对比度。 </li><li>保持期间记录在房间恒定的环境条件。调暗房间不打扰幼虫定向光。 </li></ol> 6.可选：调整FIM成像设置了录音（温度逐渐刺激条件下） 注：热梯度设备是42×42×0.2cm的3铝板带亚光黑色涂料，绝缘材料上的顶位。该板被灌注从连接到两端热能转换/冷却器和油泵两个不同的电路水（参见图1B）。温度可调节从-5到+50°C。 <ol style="margin-left: 40px;"><li>开启前的实验热梯度设备1小时，并将其放置在设置上述允许建立所希望的温度分布和组分达到平衡。 </li><li>传输与盐屏障，设置爬行表面琼脂。 </li><li>将散热板在琼脂和调整板和爬行表面到2mm之间的间距（约1毫米以上的幼虫）。 </li><li>建立的线性梯度至少0.8℃，从34℃（约2厘米的视场屏障）至18℃（约2厘米处的视场的相对部位的阻挡），通过调整/厘米温度的水回路到1°C和45°C的含量。 注：在金属板的不同梯度特性需要不同的温度，这需要根据经验确定。不需要在实验过程中要改变这些设置。 </li><li>在第5节调整设置。 </li><li>允许爬行表面在梯度平衡实验前20分钟。测试用高温计的温度梯度。 </li><li>继续进行第8。 </li></ol> 7. FIM成像（非条件刺激） <ol style="margin-left: 40px;"><li>用小画笔湿轻轻收集15幼虫从习惯培养皿中，并将它们转移到跟踪表面的中心。不转让食物仍然还是太多了水（见图7.8）。 </li><li>记录1 - 50幼虫上一个22厘米×22厘米跟踪区域。使用每个视频15只动物在大多数检查应用程序（统计原因，每个基因型使用至少100人）。 </li><li>轻轻分开的幼虫，等待约10-20秒，直到所有的幼虫开始录制前直移动。 </li><li>在每秒10帧用于非刺激条件下记录幼虫运动2分钟。使用未压缩TIF图像作为输出格式。 </li><li>在录制过程中，收集幼虫从小瓶使其习惯他们的下一个视频关键：不要打扰记录幼虫光。 </li><li>录制完成后，取出幼虫用大画笔，并丢弃它们按照当地的安全和法律规则。 </li><li>取出幼虫后，用画笔来清洁和滋润爬行表面用超纯水去离子水。 </li><li> 关键：保持表面湿润在任何时候，但避免过度莫isture，它可以被看作是晕或周围的幼虫在录音滴和干扰跟踪。 </li></ol> 8.可选：FIM成像（温度逐渐刺激条件下） <ol style="margin-left: 40px;"><li>后的温度梯度的关系成立（参见第6节），将幼虫上爬行表面（见8.2） 如后准备用于即时在20秒内记录在记录软件，设置每个视频帧的数目与定义了保存通路。 </li><li>略微抬起散热板，并放置幼虫在33℃2厘米从盐屏障。是指6和7作进一步的一般说明。再次降低了散热片，并开始录制，直接3-4分钟后，幼虫开始直线移动。 </li><li>录制完成后，直接删除的幼虫，清洁和滋润表面。请勿触摸盐琼脂，以避免氯化钠蔓延。 </li><li> 关键：保持表面湿润在任何时候，但要避免过多的水分，这可以被视为光晕或周围的幼虫在录音滴和干扰跟踪。 </li><li>使琼脂表面保湿1-2分钟后再次达到平衡，同时保存图像和收集新的动物准备下一个视频前。用高温计每5个视频控制温度梯度和（关于水的温度，高度和XY方向向跟踪表面）根据需要调整温度的设备。 </li></ol> 9.跟踪幼虫移动模式注：欲了解更多详情，请参阅附带的说明书FIMTrack（补充）。对于一个程序流程图见图2。 <ol style="margin-left: 40px;"><li>调整跟踪参数用于使用预览选项的相应的实验。 <ol><li>根据照相机和视场调整每厘米像素。 </li><li>调整基于所述摄像头设置每秒帧。 </li><li>调整亮度阈值，以便日在所有的动物都正确地检测（反馈给出在预览）。 </li><li>幼虫调整区域大小阈值。注意在预览反馈选项：单动物以黄色突出显示，碰撞幼虫以红色突出显示，每个动物的区域给出蓝色。 </li></ol></li><li>开始使用按钮在右下角跟踪。 注：成功跟踪之后，图像配幼虫轨道和含有所计算出的运动和姿态的功能csv文件被存储在图像目录下。 </li><li>使用FIM结果查看器模块（编辑&gt;结果查看器...），审查和手动调整的跟踪结果。如果需要的话，定义刺激区域评估数据中相对于所述刺激政权如爬行取向在相对 ​​于热梯度或距离到臭气物质的来源。对于更详细的说明，请使用手动（补充）。 </li></ol>数据的评估10 <ol style="margin-left: 40px;"><li>导入CSV文件导入Excel，MATLAB或任何其他程序进行进一步的统计分析。 </li></ol>

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在行为神经科学，它是强制性定量破译复杂的行为特征。需要进行统计分析。因此，大量的个体必须在高分辨率观察和自动程序。这里，FIM成像被描述，一种新颖的，简单的和鲁棒的成像设置，它提供了监测多种动物的运动的手段。利用果蝇幼虫，涡虫扁形虫和C.在FIM成像设置的有效性进行了测试线虫蠕虫。在FIM技术提供固有高对比度，以检测内部连动物的结构，如脑，气管，肠道或腺胃。重要的是，这些内部结构被鲁棒地识别，以便它们能用于自动识别所述动物19的取向。 的电影的质量可以通过水对爬行表面过量的影响。因此，关键控制琼脂的水分。表面上太旧琼脂或过多的水会导致文物。同样地，确保没有气泡被包括在抓取表面。在一般情况下，一个精心准备的琼脂表面允许录制动画4小时。 由于底层的物理原理FIM成像产生几乎无噪音的图像记录，导致卓越的图像质量。这又便于随后的基于计算机的图像分析和实现高吞吐量。然而，该方法仅限于分析动物直接接触琼脂表面。跟踪软件是由动物形成环形形状的挑战。虽然一个二进制指示符识别的环形形状，一个错误的脊柱可能被计算出。 由于跟踪表双重和三重彩色成像的模块化结构为范围。此外，额外的刺激（光，气味，电气或机械刺激）可以很容易地删除从上面ivered。该FIMTrack程序设计，以配合FIM成像的功率可容易地通过跟踪果蝇幼虫，C。线虫或涡虫。由此，并且由于其简单的和廉价的结构（见http://FIM.uni-muenster.de），FIM成像是用于广泛的生物医学应用可行，特别是允许急需高通量研究。

大多数动物不得不在一个高度成熟和可控的方式移动的能力。破译遗传基础底层运动控制它是强制性的定量评估不同的行为模式。在这方面，果蝇可作为理想模型。自由飞行果蝇的跟踪是诱人的1-4但果蝇幼虫在相对 ​​低的速度爬行发生在两个维度，因此可以很容易地监测。基于摄像头的设置结合适当的照明是用来采集图像5。这两个事件或透射光受雇于行为实验6,7。然而，由于在幼虫和幼虫运动的爬行表面忠实记录的可能光反射的半透明体可以是具有挑战性的。为了克服这些问题，一些复杂的方法已被设计出来。最近，暗视场照明被引入，以提高前景/背景续RAST 8。作为替代基于相机的记录，透镜较少的光学成像和图像传感器较少片上采集技术已经引入9-11。 几个跟踪项目最近已经推出，其中包括商业软件12和定制解决方案。高通量的跟踪程序的例子是多蠕虫跟踪（MWT）13 Multianimal步态和跟踪（MAGAT）8。两者都在共同的，即多种动物可以跟踪在一个单一的旷场舞台，使得碰撞动物导致多个新的动物的身份。为了克服此限制，一个多井设置导入分离12动物进入各个孔14。单一个体的运动的精确定量可以通过使用一个可移动的跟踪阶段结合用显微镜15来实现。然而，所有这些方法要么成本效率不高，缺乏足够的重溶液或太费时高通量的表型。 为了克服上述局限性，我们已经开发FIM（ ​​基于FTIR的成像方法）根据受抑全内反射（FTIR）16（ 图1）。这种新的成像方法提供了前所未有的高对比度，甚至允许爬行动物16的多色记录。这种方便和有效的方法的基本原理很简单。丙烯酸玻璃板上充斥着光（ 例如，875纳米红外线）。由于丙烯酸类玻璃和空气的折射率不同，则光被完全在玻璃/空气界面处反射。丙烯酸类玻璃的无加热注意到16。仅当具有较高折射率的物体触摸光线充足的表，可以点亮输入这些对象。如果动物触摸表面，光被反射，并且可以从下面（ 图1）捕获。其结果，只使触头动物的区域表现为一个亮点，它可以让影像细节与整体的黑色背景。因此，FIM-成像可以录制完美的电影的计算机视觉算法。简单和坚固的使用FIM现在带来的复杂的动物行为的详细的高通量分析成范围，可用于研究的信息处理： 例如，嗅觉8,16;远景17或18 thermosensation。 <img alt="图1" fo:content-width="6.5in" src="/files/ftp_upload/52207/52207fig1highres.jpg" width="650" /> 图1. FIM安装热刺激一体化和基本物理原理。 （A）的FIM设置。照明强度可以在前面板上进行调整。（B）提供一个热的刺激，一个黑色涂漆铝板，两边灌注热水和冷水，放在上面的琼脂表面2毫米其中本身为2毫米厚。梯度被建立在散热器板和琼脂由温度差（℃）受抑全内反射的物理原理：一个有机玻璃板由红外光照射。 θ1，θ2，θ3表示的光的反射角度。 N A，N 1，N 2和N 3表示的空气，亚克力玻璃，琼脂和幼虫的折射率分别履行不平等N A &lt;N 1 &lt;N 2 &lt;N 3。由于折射，反射角度转变过程中发生变化。如果角度小于临界角，光不再反映，可以通过图层，可以从下面拍摄的。 <a href="https://www.jove.com/files/ftp_upload/52207/52207fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在SP过程ectrum可由FIM被分析广阔。没有任何进一步的调整，FIM成像可用于监测果蝇（ 图5B）的所有幼虫阶段或可用于遵循足部印痕成年果蝇19。 C的同样，轨迹线虫或涡扁虫的运动可以很容易地记录（ 图5C）。真菌菌丝或根毛生长甚至分析似乎可行的19。在我们当前的FIM设置，4×16的红外发光二极管（IR-LED）的被集成到一个32×32 cm 2的丙烯酸系的玻璃板，被称为跟踪表（ 图1）。对IR-LED的强度取决于在跟踪表中的对象，这可以通过经由脉宽调制（PWM），其连接到所述电路微控制器来容易地进行的重量进行调整。 FIM产生非常高的对比度的图像在宽范围的照明强度。重要的是，它根erates在已经很低的总体红外irridation了优异的成绩。 用红外滤波器的摄像机放置在跟踪表，它允许集成的额外刺激到设置在下面。热刺激可以通过热辐射板可以很容易地应用和光刺激被由LCD投影机施加。还加臭剂可以通过简单的盖子8被包含在梯度。对热梯度的实验中，散热板被分别灌注用热水和冷水在两侧，并放置幼虫（ 图1B）超过2毫米。 的高对比度，高品质的电影的产生将打开的可能性为复杂的计算机图像分析，从而我们实施了FIMTrack软件来提取一大组的特征，从图像（ 图2）。前六个主要特征是从动物（ 图3A）的轮廓限定。这些功能提供基线为进一步的计算描述该动物的形状和其在某些刺激位置在给定的时间点（ 图3B）6次要特征。目前，九三级功能是计算正在将时间方面，从而与主要和次要特征（ 图3C）的动物的运动特征在一起。 <img alt="图2" fo:content-width="6.5in" src="/files/ftp_upload/52207/52207fig2highres.jpg" width="650" /> 图2. FIMTrack概述，算法的工作流程和幼虫表示。 （A）如何使用FIMTrack。图像被加载。灰度值阈值和幼虫大小阈值定义单一的幼虫必须设置。幼虫面积必须在[分钟尺寸，最大尺寸。跟踪是通过突出显示的按钮启动。（B）跟踪的工作流程。被点击开始按钮后，背景图象为Calculated（最小强度随着时间的推移）。只要有左帧，幼虫是基于灰度阈值和min-和最大尺寸的阈值分割。对于幼虫表示计算所有分段（比较（C））。每一个新的模型被关联到一个给定的轨迹，如果一个有效的轨道是可用的。如果最后帧为止，最后完成后处理完成后的输出的生成。（C）的幼虫表示。动物包括一个头部和尾部点（H和T）的。这些点之间的脊点秒的任意奇数号i可以被设置具有半径R I。此外，质量为m和主体弯曲角度γ的中心被计算出来。几种运动相关的参数是由紫色的线条勾勒。 <a href="https://www.jove.com/files/ftp_upload/52207/52207fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图由FIMTrack计算3.特点。 （A）主要功能基于动物的轮廓上。（B）二次特点的基础上，主要功能。（C）第三功能的基础上，在连续帧的和额外的投入主要功能<a href="https://www.jove.com/files/ftp_upload/52207/52207fig3highres.jpg" target="_blank">，请点击这里查看这是一个更大的版本数字。</a>

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">Name of the Material/Equipment</td>
			<td style="width:191px;">Source</td>
			<td style="width:191px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">FIM setup</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">details for construction or purchase of setups is available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrylic glass plate</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">Additional for agar pouring</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Heat radiator plate</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">Aluminum plate (paintet in matt black) perfusable on opposing sites with adjustable mounting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:191px;">Water calorifier/cooling pumps and hoses</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">based on GE healthcare MultiTempIII (No.: 18-1102-78) and Dr Bruno Lange GmBH (Typ: LTG013)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Standard Camera (4 MP)</td>
			<td style="width:191px;">Basler</td>
			<td style="width:191px;">acA2040-25gm</td>
			<td style="width:251px;">Camera defaultly used for the FIM setup</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Test Camera (1.4 MP)</td>
			<td style="width:191px;">QImaging</td>
			<td style="width:191px;">&#160;1394 firewire (01- QIC-F-M-12 MONO)</td>
			<td style="width:251px;">Camera used for comparison</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Test Camera (0.8 MP)</td>
			<td style="width:191px;">Point Grey</td>
			<td style="width:191px;">Dragonfly 2 (DR2-13S2M/C-CS)</td>
			<td style="width:251px;">Camera used for comparison</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test Camera (0.3 MP)</td>
			<td style="width:191px;">Sony</td>
			<td style="width:191px;">PS Eye USB2.0 camera</td>
			<td style="width:251px;">Camera used for comparison</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:191px;">Computer</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;">equipped with at least i5 Intel processor or better, 16 GB RAM and sufficient HDD storage space [&#62;1TB]</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Standard Fly food</td>
			<td style="width:191px;">Custom&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">Standard Fly vials 135 ml</td>
			<td style="width:191px;">Sarstedt AG&#38;Co, N&#252;mbrecht, Germany</td>
			<td style="width:191px;">78,895</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Petri dishes 9cm</td>
			<td style="width:191px;">Sarstedt AG&#38;Co, N&#252;mbrecht, Germany</td>
			<td style="width:191px;">821,473</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ultrapure deionized water</td>
			<td style="width:191px;">Merck Millipore, Darmstadt, Germany</td>
			<td style="width:191px;">Synergy&#160;</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaCl</td>
			<td style="width:191px;">Carl Roth GmbH, Karlsruhe, Germany</td>
			<td style="width:191px;">3957.2</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Food grade agar</td>
			<td style="width:191px;">AppliChem GmbH, Darmstadt, Germany</td>
			<td style="width:191px;">A0917,5000</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paintbrush (small and large)</td>
			<td style="width:191px;">Milan</td>
			<td style="width:191px;">Aquarell 310 Size 0 and 2</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Pyrometer</td>
			<td style="width:191px;">Trotec</td>
			<td style="width:191px;">BP20</td>
			<td style="width:251px;"></td>
		</tr>
	</tbody>
</table>

fim imaging and fimtrack: two new tools allowing high-throughput and cost effective locomotion analysis

神经元网络功能的分析需要的行为特征的可靠测量。由于自由活动动物的行为是可变到一定程度，许多动物都进行分析，以获得统计学显著数据。这又需要的运动模式的计算机辅助自动定量。以获得几乎半透明和小型移动物体的高对比度图像，基于受抑全内反射称为FIM一种新颖的成像技术被开发。在此设置中，将动物仅在与下面的爬行表面接触的非常具体的位置照射红外光。这种方法导致了非常高的对比度的图像。随后，这些高对比度图像是使用建立轮廓跟踪算法处理。在此基础上，我们开发了FIMTrack软件，其用于提取数的所需特性来定量描述了大量的各种运动的特点。在这个软件包的发展，我们专注于一个开源的架构，允许轻松添加更多的模块我们的努力。程序的操作独立于平台，并伴随着一个直观的图形用户界面引导通过数据分析该用户。所有的运动参数值给出了CSV文件，允许进一步的数据分析形式。此外，结果查看器集成到跟踪软件提供了机会，交互审查并调整输出，可能在刺激整合是必要的。 FIM和FIMTrack的动力表现在研究果蝇幼虫的运动。

用于成像的几个不同的照相机具有不同的分辨率的性能进行了测试（图4）。所有的摄像机，其中配备了适当的IR滤镜。根据在该试验中，价格最低的照相机的分辨率低，视场限制为10厘米×10厘米。最好的结果是使用4兆像素（MP）相机获得。这导致每个第三龄幼虫长度为100像素的分辨率，并允许容易地识别内部结构。此外，动物的蠕动能够容易地提取出（ 图4A）。然而，人们可以使用较便宜的照相机，它也可以由FIMTrack分析仍然获得高对比度的电影。使用1.4 MP照相机与深度的8位和1392 X 1040像素的分辨率大约是一半的价格，并允许每三龄幼虫长度45个像素中的视场的分辨率。头，但没有其他的内部结构可以被识别（ 图4B）。跟踪和检测的蠕动是可能的，但精度降低（ 图4B）。 用更便宜的0.8 MP相机的空间分辨率堪比1.4万像素摄像头，幼虫头部不能再忠实地承认（ 图4C）。跟踪和蠕动的分析是可能的，但包括基础上增加了更多的噪音抖动。出人意料的是，即使是低分辨率的USB摄像头提供了足够的高质量的电影来计算幼虫轨迹（0.3万像素摄像头，低于20€， 图4D）。该蠕动可从面积计算，但测量非常嘈杂。 在我们的设置中，我们经常使用的400万像素摄像头。用于筛选，这种相机可监听一个22厘米×22厘米竞技场，这显然提供了机会，同时分析大量的动物，使得高通量分析是可行的。使用此设置，幼虫长度i177。40像素仍然可录制和蠕动的分析表示。 15幼虫轨迹中的热梯度的示例性图像是在图5A中 。此外，利用微距镜头的允许图像幼虫具有非常高的分辨率，其中许多内部器官变得可见，头识别性进一步提高（ 图5B）。此外，这些可用于大范围的行为20的进一步更详细的分析。相同的设置可以方便地用于图像抓取C.线虫蠕虫（ 图5C）。 <img alt="图4" fo:content-width="6.5in" src="/files/ftp_upload/52207/52207fig4highres.jpg" width="650" /> 图4. FIM成像和跟踪结果不同的相机。 （A）左图：3幼虫爬行使用400万像素摄像头，支持10帧拍摄的10厘米×10厘米跟踪阶段FIM成像。中秋节：剪裁和一个幼虫大规模轨迹中心。动物的区域被指示。右：幼虫面积绘制了100帧。红色箭头表示截取图像的时间点。（B）相当于（A），但使用的是1.4万像素摄像头拍摄的。（C）相当于（A），但使用的是0.8万像素摄像头拍摄的。（D）相当于（ A），但使用的是0.3 MP相机拍摄。 <a href="https://www.jove.com/files/ftp_upload/52207/52207fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" fo:content-width="6.5in" src="/files/ftp_upload/52207/52207fig5highres.jpg" width="650" /> 图5.热刺激，和高分辨率的应用程序。 （A）热的刺激的应用（比较图1）。通过FI计算的轨迹MTrack。（B）中使用的是微距镜头的第三，第二和第一龄幼虫的高分辨率图像应用。由400个像素中的视图的2.5×2.5厘米的场（c）一种 C的第三龄幼虫的长度来表示。使用FIM成像线虫蠕虫抓获。比例尺表示。 <a href="https://www.jove.com/files/ftp_upload/52207/52207fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis at JoVE.com

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

FIM是旨在跟踪小型移动物体如C一种新型的，具有成本效益的成像系统线虫，涡虫和果蝇幼虫。伴随FIMTrack计划的目的是提供快速，高效的数据分析。总之，这些工具允许的行为特征高通量分析。

FIM成像和FIMtrack：两个新的工具允许高吞吐量和成本效益分析步态

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

fim-imaging-fimtrack-two-new-tools-allowing-high-throughput-cost

Research

JoVE Journal

Neuroscience

11.6K 次观看.  Westfälische Wilhelms-Universität Münster.  FIM是旨在跟踪小型移动物体如C一种新型的，具有成本效益的成像系统线虫，涡虫和果蝇幼虫。伴随FIMTrack计划的目的是提供快速，高效的数据分析。总之，这些工具允许的行为特征高通量分析。 

视频: FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

同时神经刺激，并记录在水迷宫的设计和构建一个具有成本效益探头

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

在斑马鱼幼虫的高通量自动化的行为分析

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

受体激活的测量通过荧光素酶活性：哺乳动物的嗅觉受体的高通量分析

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

两种算法的高通量和多参数定量<em&gt;果蝇</em&gt;神经肌肉接头形态

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

果蝇岛试验中运动行为的高通量分析

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

高通量图像引导立体定向神经导航和聚焦超声系统，用于在啮齿动物中打开血脑屏障

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...