Name: preparation and 3d tracking of catalytic swimming devices
Uploaded: 2016-07-01T15:00:00.000+00:00
Duration: 6 min 50 s
Description: Watch this Scientific Journal Video about Preparation and 3D Tracking of Catalytic Swimming Devices at JoVE.com

This work was supported by EPSRC Career Acceleration grant EP/J002402/1.

注意:使用前请咨询所有相关的材料安全数据表。在这个协议中使用的过氧化氢是有害的，并且在暴露于铂氧气的演进带来爆炸危险。该协议包括工程控制，同时处理过氧化物溶液（通风柜）和个人防护设备（防护眼镜，手套和白大褂）过程中使用的所有适当的安全控制。 1.使催化Janus粒子<ol><li> 准备搏命基板 <ol><li>使用顺序洗涤每个在去离子（DI）水，玻璃去污溶液和DI水几分钟清洁的未使用标准显微镜载玻片。乙醇V混合物:然后用70:30 v洗去离子水，最后在一个干净的空气/氮气流吹干。 </li><li>在显微镜下检查载玻片，以验证该表面距微粒污染的证据清洁无。重复步骤1.1。1，如果需要的。 </li></ol></li><li> 准备沉积胶体分散体系 <ol><li>吸移管将10μl水溶液库存荧光胶体溶液（10％重量）放入990微升乙醇。根据用于到达约0.1％（重量）的胶体悬浮液原液浓度根据需要调节卷。 </li><li>涡旋混合10秒。 </li></ol></li><li> 旋涂胶体分散到载玻片基板 <ol><li>装备用干净的玻片上旋涂机。填用100μl上面制备的稀释胶体溶液的吸移管尖。程序旋涂机以执行30秒，2000转周期。开始旋涂机，并且当加快速度连续吸取所述制备的溶液到纺丝载玻片的中心。 </li><li>从旋转涂布机中取出载玻片，回到光学显微镜和验证甚至主要是非接触不同的胶体分散覆盖中部地区ÒF中的搏命。 </li></ol></li><li> 真空蒸发铂金属胶体上装饰载玻片 <ol><li>插入胶体涂覆载玻片为金​​属蒸发器。确保胶体装饰侧朝向蒸发源。安装的铂金属蒸发源和沉积铂金属的15纳米。 注意:下面的金属沉积，样品应当存放在惰性气氛下。 </li></ol></li></ol> 2."游泳"Janus粒子<ol><li> 重新悬浮的Janus胶体成含有过氧化溶液 <ol><li>切割晶状体组织中的1×1cm的正方形，挫伤与10微升的去离子水结束。用镊子保持，并沿镀铂胶体装饰玻璃载玻片表面轻轻揉搓（此步骤在物理上从衬底移除胶体）。 </li><li>将镜头纸到1.5毫升去离子水，大力摇晃手动30秒在一个密封的涂是。取下镜头组织。 </li><li>吸管1ml含有溶液到新的容器中的胶体，填充用1ml 30％w / v的H 2 O 2的储液。轻轻混匀该溶液，然后将在室温下超声浴中5分钟，随后再25分钟未搅拌潜伏期。 注意:此解决方案可能演变的氧气;不密封。 </li><li>干燥的100微升剩余的含水胶体溶液到的扫描电子显微镜（SEM）存根使的Janus胶体结构的SEM验证。14 </li></ol></li><li> 准备分析比色皿 <ol><li>额外1毫升DI水添加到含有过氧化和镀铂胶体，以在适当的燃料强度（10％）到达，以允许快速推进的温育溶液。 注:现有孵化阶段是在浓度较高的燃料浓度的清洁铂催化剂表面进行。 </li><li>补罗W¯¯量矩形石英玻璃比色皿与培养方案。松配合推的帽子。 注意:爆炸危险 - 不使用螺丝帽。 </li></ol></li></ol> 3.显微观察<ol><li> 定位感兴趣的颗粒 <ol><li>负载反应杯到装有合适的目标（ 例如，20X）的荧光显微镜和使用的过滤器的适当组合（激发450-490纳米，发射&gt; 515纳米）激发荧光团的发射。 </li><li>手动搜索的反应杯内的荧光胶体。 注意:调节胶体密度，同时保持过氧化物浓度可能需要。例如，推荐的稀释如果胶体密度高，并产生流过大量的氧气气泡存在。约0.003％胶体体积浓度是建议起点。 </li><li>优化3D追踪光学设置:合适的光照条件下，我N对焦胶体会出现尖锐圆形物体。然而，由于推进器移动胶体进出焦平面的独特的尺寸变更周围将观察到球体中心的亮环，这是用来确定的z坐标，以使3D跟踪。 </li></ol></li><li> 录制视频 <ol><li>开始视频捕捉之前，聚焦显微镜，使得所关注的粒子产生的同心环，与颗粒"下"的聚焦位置。在视频拍摄不移动焦点平面。 </li><li>感兴趣的颗粒录制视频。使用带有超过30赫兹帧频30秒的视频时长，以便详细的弹道重建。 </li></ol></li><li> 三维轨迹重建 <ol><li> 校准Z轴 <ol><li>补2％（重量）。在60℃下，放置在含有悬浮荧光Janus粒子的水吉兰糖胶溶液中的等效反应杯至以上使用，并且允许设置以形成含有固定静态胶体刚性透明胶凝样本。 </li><li>着眼于使用上述选择的相同照明条件下单个固定胶体，现在记录一系列静止图像作为z轴焦点是通过相对于这个平面已知位移上升。 </li><li>确定在每个已知焦点位置11的环的半径。 注意:这是最有效地使用可作为间歇方法被应用到所有的校准的图像分析算法来执行静止帧和视频。一个典型的方法涉及平滑图像，阈值，以确定所述对象的中心的近似位置，然后通过测量强度峰任环的侧面之间的距离定位的实际x和环中心的y坐标。从环中心的强度的峰的平均径向距离然后可以发现11这使得亮环的两个半径D中的x​​y坐标与子像素精度来确定。通过定位的x，y和z从焦平面固定在绞兰胶30μm的两面神球体的位置，在图像的时间序列，粒子可位于与沿每个轴±25纳米的误差。该错误可以归因于在图像噪声。噪比并因此信号，的位置的算法的准确度依赖于检测到的荧光的光的强度。当的Janus球体远离焦平面其强度变得太弱准确跟踪它， 例如 ，对于一个直径4.8微米的胶体的约200的Z范围微米是可能的。另一种非算法的方法是使用X，Y中心和半径的简单手工测量，然而，这将降低准确性。 </li><li>绘制校准曲线，以涉及半径z-位置，并适合于适当的功能（ 例如 ，三次方程），以允许内插。11 </li></ol></li> &lt;李&gt; 校准X，Y轴 <ol><li>记录使用3.2选择了相同的显微镜条件下的静止画面的空间标定刻度的光学显微镜图像。 </li><li>测量与已知的真实世界大小的物体从空间校准刻度图像的"像素"的尺寸和使用它来建立一个像素微米的转换系数为X，Y图像平面。 </li></ol></li><li> 弹道重建 <ol><li>确定所述视频序列的每一帧中的x和y坐标和半径如3.3.1.3中所述，使用在3.3.1.4发现半径转换成z中的功能，和在3.3.2.2中找到的校准系数来转换x和y像素坐标到微米。此过程将导致在一个准确的x，y，z坐标的推进颗粒位置作为时间的函数。11本步骤可使用算法手动来实现，或。 </li><li>确定衍生properties诸如平均速度量化观察催化游泳的程度。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

在铂Janus粒子制备协议的许多因素会影响观测到的轨迹。由于使用2微米的直径的颗粒会给推进速度在每秒10微米量级描述的参数。如果使用更小的颗粒，速度会增加，同时增加粒度会降低推进速度。12蒸发协议的细节也将改变所观察到的轨迹。在当前的协议，推荐胶体的稀疏分布，以金属蒸镀垂直于滑动方向在一起。这些条件导致如在图2中，这导致布朗旋转扩散的限制范围内的线性轨迹中所示的对称的Janus结构。13相反，如果紧填充胶体受到掠射角沉积，则实现Janus帽的对称性可破，诱使旋转行为。14霸这里出产的rticles显示在所有三个维度相对各向同性的议案;然而，如果使用更厚的铂涂层，或更大的颗粒中，向上偏压或gravitaxis可以赋予。制造后的Janus胶体的存储的11详细还可以影响所观察到的游泳速度。高表面能清洁铂表面从蒸发阶段新兴易受表面污染从例如烃，特别是硫醇。15 此外，该溶液的性质，其中的Janus胶体再悬浮是用于观察推进的关键。低过氧化物浓度将导致较慢的速度，作为分解反应产生的运动的速率减少。6此外，低浓度的盐会造成在推进速度的显着降低的。7 这里生产的胶体的一个主要特点是他们的NEutral浮力，这使得它们适合3D跟踪。一般游泳装置的领域已很少关注3D效果，部分原因是由于从致密金属正在作出一些突出的例子，使它们迅速沉淀，16而且还由于与使所需的测量有关的困难和费用。对于一些建立三维跟踪方法明显的缺点，这些快速移动胶体存在，例如，共焦激光扫描显微镜可缺少的时间分辨率来记录足够数量的图像来解决轨迹。在这种情况下，我们在这里提出的方法具有只需要一个单一的框架，以允许z坐标的估计，这因此允许高帧速率的显著优势。另外，作为Z坐标的重建仅仅依赖于外的焦点的胶体在单个帧的相对对比度，而不是绝对的荧光强度，它是弹性的淬火和闪烁的效果在荧光团。这些优点是有可能在场的减少的深度在其上的3D轨迹重建是可能的费用，以及用于很好地分离非重叠胶体的要求。我们希望，描述协议将允许在3D行为的其他感兴趣的研究组供游泳设备直截了当和具有高度的精度访问此信息。很显然，扩展这些设备3D的理解将开辟性的未来的现象和应用显著范围。感兴趣的轨迹分析的进一步细节读者引向参考17，描述了在推进系统，以及如何确保推进速度的精确量化的公共构件。

催化游泳设备是小规模，能够在流体环境下自主生成运动不受限制胶体1,2这些设备正在吸引显著的研究兴趣，因为他们必须启用激动人心的新功能，如药物输送，3实验室芯片上的运输潜力4和环境整治。5一个被广泛研究的例子是催化"剑锋"游泳者。6这些粒子由两种不同的两侧，或面（剑锋是一个两面对罗马神）得到他们的名字。一面是催化活性的，并且能够进行分解反应，而另一种是惰性的。在合适的溶解燃料分子的存在，所产生的不对称化学反应产生在其周围可以产生经由自diffusiophoresis /电泳运动的胶体梯度。7 表征为这些快速移动的物体的运动是茶<html> llenging和许多实验观察到迄今被限制在二维。然而，最终的应用很可能利用催化游泳设备在整个大宗解决方案在3D能力。8要解决这个问题，在这里我们描述了一个协议，允许被确定游泳设备精确的三维轨迹。此方法是基于解释由出具有固定焦点物镜，9观察焦点荧光胶体制备的环结构，并且易于使用常规未修改显微镜适用。这里所描述清楚这种方法，在这一领域的其他研究人员将能够访问这些3D信息中获益。这将有助于未来的见解运动特性游泳设备。这种潜在的证据是由最近的游泳设备报告给被定向在重力作用下，10,11行为，可以非常容易地通过3D追踪的应用可视化。11 ove_content"&gt;本文还清楚地记录了制造催化剑锋颗粒游泳设备，这将是进一步受益规范跨越现有的研究小组调查这些设备的方法，并且还引导热衷于制造和调查游泳设备的新的研究方法。

<table><tbody><tr><td>Evaporator</td><td>Moorfield (UK)</td><td></td><td>Minilab 80 e-beam evaporator</td></tr><tr><td>Microscope</td><td>Nikon</td><td></td><td>Eclipse LV100</td></tr><tr><td>Fluorescence light source</td><td>Nikon</td><td></td><td>Nikon B2A filter cube</td></tr><tr><td>Objective</td><td>Nikon</td><td></td><td>20X, 0.45 NA</td></tr><tr><td>Cuvette</td><td>Hellma</td><td></td><td>fused quartz, 40 x 10 x 1 mm</td></tr><tr><td>Vortex mixer</td><td>IKA</td><td></td><td>Lab Dancer S2</td></tr><tr><td>Spin coater</td><td>Laurell Technologies Corp.</td><td></td><td>Model WS-400BZ-6NPP/Lite</td></tr><tr><td>Ultrasonic bath</td><td>Eumax</td><td></td><td>2 liter</td></tr><tr><td>Lens tissue</td><td>Whatman</td><td>2105 841</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma-Aldrich</td><td>31642-1L</td><td>30 wt%</td></tr><tr><td>Platinum</td><td>Sigma-Aldrich</td><td>267171</td><td>0.25 mm, 99.99%</td></tr><tr><td>Colloids</td><td>Thermo Scientific</td><td></td><td>Fluoro-Max PS microspheres, d = 1.9&amp;nbsp;microns</td></tr><tr><td>Glass decontamination solution</td><td>Fisher Scientific</td><td>D/0025/15</td><td>Decon 90</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>E/0600DF/17</td><td>Absolute Ethanol</td></tr><tr><td>DI water</td><td>Elga</td><td></td><td>Purelab Option filtration system (15 MW)</td></tr><tr><td>Gellan gum</td><td>Sigma-Aldrich</td><td>P8169-100G</td><td>&quot;Phytagel&quot;</td></tr></tbody></table>

preparation and 3d tracking of catalytic swimming devices

We report a method to prepare catalytically active Janus colloids that "swim" in fluids and describe how to determine their 3D motion using fluorescence microscopy. One commonly deployed method for catalytically active colloids to produce enhanced motion is via an asymmetrical distribution of catalyst. Here this is achieved by spin coating a dispersed layer of fluorescent polymeric colloids onto a flat planar substrate, and then using directional platinum vapor deposition to half coat the exposed colloid surface, making a two faced "Janus" structure. The Janus colloids are then re-suspended from the planar substrate into an aqueous solution containing hydrogen peroxide. Hydrogen peroxide serves as a fuel for the platinum catalyst, which is decomposed into water and oxygen, but only on one side of the colloid. The asymmetry results in gradients that produce enhanced motion, or "swimming". A fluorescence microscope, together with a video camera is used to record the motion of individual colloids. The center of the fluorescent emission is found using image analysis to provide an x and y coordinate for each frame of the video. While keeping the microscope focal position fixed, the fluorescence emission from the colloid produces a characteristic concentric ring pattern which is subject to image analysis to determine the particles relative z position. In this way 3D trajectories for the swimming colloid are obtained, allowing swimming velocity to be accurately measured, and physical phenomena such as gravitaxis, which may bias the colloids motion to be detected.

图1示出胶体上之前沉积铂干净的载玻片上的典型的分散; 图2示出了用于涂敷的Janus游泳半铂典型背散射SEM图像，在该成像模式的铂涂覆的区域产生了鲜明的对比。所需的半球形铂层是显而易见的。 图3显示了典型的荧光的Janus游泳者的固定在绞兰胶最佳照明条件下的外观。游泳者显示为对称的环的功能，而且它是一个可以被用来确定所述胶体相对于聚焦位置的z位置的环的半径。 图4示出了代表的横截面的径向亮度强度分布在组合使用图像分析算法来准确定位中心和胶体的表观半径。 图5 </stron<html> g&gt;的包含使用一个固定的胶体样品和校准显微镜z轴平移阶段涉及从焦点位置表观胶体尺寸和距离而获得的校正曲线。此曲线被装配到一个三次函数，这是用来表观半径转换为z坐标。最后， 图6显示一个典型的x，y，为荧光的Janus粒子游泳ž轨迹。 <img alt="图1" src="/files/ftp_upload/54247/54247fig1.jpg" /> 图1. 直径1.9微米的聚苯乙烯微光学图像。微球分散在铂金沉积之前清洁的载玻片。比例尺代表40微米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ES / ftp_upload / 54247 / 54247fig2.jpg"/&gt; 图2. 直径为1.9微米的聚苯乙烯微球的SEM背散射图像。在微铂沉积后显示。比例尺代表2微米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54247/54247fig3.jpg" /> 图3. 固定在使用20X物镜（0.4 NA）的记录，吉兰糖胶的直径4.8微米的荧光聚苯乙烯球的校准图像。每个图像下面的距离表明球体上述物镜的焦平面的距离。作为图像从0到200微米的一个明亮的光盘变为亮环的聚焦图像散焦，在半径为依赖于放大率时，spherE尺寸和焦平面的距离。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54247/54247fig4.jpg" /> 图4 的X，Y，Z粒子跟踪过程。一组自写的算法被用来通过提取一系列垂直线和水平线，并找到平均值中期首先找到亮环的（X，Y）中心亮峰之间的点（A）。环半径然后从安装到平均像素灰度值从环中心（B型）辐射出样条曲线的峰值强度计算。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5的Z坐标校准图表用于通过测量固定在绞兰胶球的亮环的半径获得的Janus球体（参见图3和4），校准图表用于由我们的算法，以测得的环半径转换为Z-坐标。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54247/54247fig6.jpg" /> 图6. 典型荧光的Janus球体游泳装置的轨迹。移动游泳装置的图像的序列在33赫兹的帧频被记录在一段30秒。通过定位亮环中心得到的轨迹（X，Y，Z）坐标（FI古尔图4（a））和比较测量圈半径校准图表序列中的每个图像（ 图4（b）和5）。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54247/54247fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Preparation and 3D Tracking of Catalytic Swimming Devices at JoVE.com

Preparation and 3D Tracking of Catalytic Swimming Devices

A method to prepare catalytically active Janus colloids that can "swim" in fluids and determine their 3D trajectories is presented.

催化游泳器件的制备及其三维跟踪

We report a method to prepare catalytically active Janus colloids that "swim" in fluids and describe how to determine their 3D motion using fluorescence ...

preparation-and-3d-tracking-of-catalytic-swimming-devices

Universidad de Cartagena UDEC

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Engineering

Unit 1

Motion in Two or Three Dimensions

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7.5K 次观看.  University of Sheffield. A method to prepare catalytically active Janus colloids that can "swim" in fluids and determine their 3D trajectories is presented.

视频: Preparation and 3D Tracking of Catalytic Swimming Devices

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

采用触摸诱发反应和运动测定以评估在斑马鱼肌肉性能和功能

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

科研

发育生物学

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a &#34;swim ergometer&#34; by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL&#8729;min-1&#8729;kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

游泳运动员心肺评估的快速递增的绑带游泳最大协议

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen ...

生物化学

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

3D运动性步态分析,用于啮齿动物的临床前研究

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...

行为学

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

自由游泳弱电鱼的长期行为跟踪

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

神经科学

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

心肌肌细胞基致动器和自稳定Biorobot  - 第1部分

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

生物工程

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Swimming Performance Assessment in Fishes

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.

The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

鱼类游泳绩效评估

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, ...

生物学

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

的自动分析<em&gt;℃。线虫</em&gt;游泳行为使用天书软件

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes&#8212;many not associated with the oral apparatus (OA)&#8212;that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

使用细胞追踪分析口腔器械再生期间和之后的张力模式

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds ...

催化游泳器件的制备及其三维跟踪

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