Name: fourier-based diffraction analysis of live caenorhabditis elegans
Uploaded: 2017-09-13T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans at JoVE.com

我们感谢胡安。我们感谢瓦萨大学本科生研究暑期研究所 (熊胆)、露西-梅纳德三文鱼研究基金和 NSF 奖 No. 1058385 的支持。

1. C. 线虫 生长和维护 <ol> <li> 准备线虫培养皿。
	<ol> <li> 用琼脂溶液填充培养皿, 然后让它们凝固, 然后用 OP50 菌株的 大肠杆菌 区域性进行播种 9 , 10 . </li>
	</ol> </li> <li> 在每个板上准备一个成年线虫的起始种群, 将几个成虫移动到新鲜琼脂填充的培养皿中, 并带有一个 大肠杆菌 补丁。保持20和 #176 的线虫培养; C 在孵化器中. 
	注意: 线虫菌株可从 线虫线虫 基因组中心获得。为这项研究, 狂放的类型, N2, 劳损和 OH7547 (otls199 [猫-2:: GFP + rgel-1 (F25B3.3)::d 失调 + 卷-6 (su1006)]) 应变, 陈列滚轮表型, 被使用了. </li>
	<li> 为未来的区域性传播蠕虫。
	<ol> <li> 将包含 C. 线虫 的 petri 盘和温度控制箱中未使用的生境培养皿移除。把它们放在解剖显微镜的舞台上. </li>
		<li> 点燃本生燃烧器, 并通过将金属放入火焰中以使其呈红色, 来杀菌铂线虫的选择。允许选择冷却到室温。不要设置拾取或让选择接触到污染物. </li>
		<li> 轻轻触碰尖端的拾取到细菌圈的边缘。这种物质是粘性的, 将使采摘个别成年线虫更容易. </li>
		<li> 将多达4的妊娠线虫转移到线虫生长培养基 (NGM) 琼脂填充的培养皿中, 并在20和 #176 孵育;蠕虫将产卵, 将在四天内成熟. </li>
		<li> 在移动四成虫线虫后, 将剩余的线虫返回孵化箱. </li>
	</ol> </li> </ol> 2。光学设置 ( 图 1 ) <ol> <li> 在光学工作台的后左角附近保护氦氖激光器, 并将其连接到电源. 
	注: 激光和 #39 的光束必须满足采样的要求。 C. 线虫 的长度约为1毫米, 因此, 在线虫发生的情况下, 激光束的直径应大于2毫米, 但不大于5毫米, 因此不难找到衍射图案. </li>
	<li> 在氦氖激光器和样品之间放置一个中性密度的过滤器, 使激光光束在到达样品之前通过过滤器进行传输. </li>
	<li> 使用两个前表面铝制转向镜, 通过在中性密度过滤器后保护第一个镜像来建立潜望镜。将第二个镜子固定在第一个镜子下面10厘米的地方, 给出空间来引导激光束, 并在镜子之间插入试管。将激光束和试管对准, 使激光束通过试管垂直移动. 
	注意: 从衍射生物体到光电二极管的距离必须比生物体本身要大得多, 才能达到远场衍射。在这个实验中, 从试管到光电二极管的距离是20厘米. </li>
	<li> 将光电二极管直接从第二个镜像中保护, 并将其传感器朝向镜像. 
	注: 含有线虫的试管将用化学钳放置在两个镜子之间。参见4和5节. </li>
	<li> 将一个充水的试管放在支架上。调整展位高度。调整镜1和镜2的高度和角度, 使激光光束通过靠近试管的方向移动, 而不是直接对准光电二极管. </li>
	<li> 使用一个级别来确保展台形成试管的平整曲面。如有必要, 请进一步调整镜像. </li>
	<li> 使用数字示波器提供的 USB 电缆将光电二极管连接到数字示波器。将数字示波器连接到将用于记录和保存数据的计算机. </li>
</ol> 3。示波器设置 <ol> <li> 使用计算机上的示波器软件, 将采样速率设置为至少 8 Hz, 以充分解决蠕虫的颠簸循环. 
	注意: 采样率应该比预期的抖动频率的两倍还多, 因此奈奎斯特定理 11 满足. </li>
</ol> 4。准备数据收集的蠕虫和试管 <ol> <li> 将四条成虫线虫转移到新鲜的 NGM 10 琼脂填充的 Petri 板, 使用薄的扁平铂线拾取 (见第1.3 节). </li>
	<li> 从包装上卸下一次性塑料试管, 小心只触及其脊边上的试管. </li>
	<li> 使用微将蒸馏水注入试管, 直到试管大约80% 的蒸馏水. 
	注: 在处理线虫时, 只有使用蒸馏水或电离缓冲器, 如 M9 10 或磷酸盐缓冲盐水 (PBS), 因为自来水中含有微生物杀死的化合物. </li>
	<li> 将含有 C. 线虫的培养皿放置在解剖范围内使用 . </li>
	<li> 使用铂金镐, 从培养皿中取出一条成熟的 C. 线虫 , 然后将其浸入试管中, 如有必要, 将其移入圆圈以去除线虫. </li>
	<li> 为了防止气泡在试管中形成, 请用水填充试管, 直到它在试管和 #39 上略微凸出。将试管和 #39 的盖子全部装满水, 然后迅速把盖子放到试管上. </li>
	<li> 使用光学清洁布除去可能溢出的水滴和光学清洁纸, 以清除任何小的残留液滴. </li>
</ol> 5。实时数据采集衍射图样强度变化 <ol> <li> 打开氦氖激光器并调整频率/颜色设置, 使其产生红色光束。打开传感器. 
	 警告: 使用低能量光束在 632 nm, 作为 C. 线虫 避免高频 (蓝色) 光 12 。
	<li> 在试管中定位蠕虫。将试管在其脊边上, 轻轻地倾斜试管直到线虫近似地位于试管部分的中心. 
	注意: 摇晃或倾斜试管猛烈地导致蠕虫与试管的墙壁碰撞。这会破坏线虫。
	<ol> <li> 将试管放置在光学系统中的支架上, 将其置于激光和 #39 中, 从镜像1反射到镜像2。一定要消除杂散光. </li>
	</ol> </li> <li> 将蠕虫集中在激光束中。
	<ol> <li> 将光电二极管置于衍射图样中, 以使光电二极管的位置和衍射图样的中心最大值不重合. </li>
		<li> 调整中性密度过滤器以防止光电二极管饱和。旋转中性密度过滤轮调节光强。
		<ol> <li> 旋转中性密度滤镜, 使光电二极管的电压输出增加. 
			注: 使用数字光电二极管的软件观察电压输出。如果电压不变, 光电二极管就会饱和。在这种情况下, 旋转中性密度的过滤器, 直到电压降低, 而不会在最小读数时变平。确保电压信号在峰值读数不平坦, 指示光电二极管的饱和度。如果观测到饱和度, 则通过旋转中性密度过滤轮来减小光强. </li>
		</ol> </li> <li> 一旦移动的衍射模式可见, 通过单击控制示波器的软件上的 "启动" 按钮来收集数据, 同时监控蠕虫和 #39 的移动。继续测量直到蠕虫从激光束和 diffrac 中移出模式消失, 通常需要大约二十年代。
		<ol> <li> 通过单击示波器软件上的 "停止" 按钮来停止数据收集过程。以. csv 或 .txt 格式保存每个试用和 #39; s 数据. </li>
		</ol> </li> </ol> </li> <li> 重复步骤 5.2-5.3, 直到至少为每个表型收集了50数据集。每次试验使用八至十只动物. </li>
	<li> 如果试管被划伤处置它和蠕虫, 并重复步骤4。如果蠕虫在转移中受到损害, 则在重复步骤4使用相同的试管之前, 将其处理并用蒸馏水冲洗试管. </li>
	<li> 重复步骤5使用 OH7547 和 #34; 轧辊和 #34; 应变, 小心标签的数据, 以表明蠕虫的应变. </li>
</ol> 6。数据的傅立叶频谱 <ol> <li> 将获得的数据导入到能够执行离散傅立叶变换的数据分析程序 3 。
	<li> 使用软件的快速傅立叶变换 (FFT) 选项对每个数据集执行傅立叶变换. </li>
	<li> N2 野生类型蠕虫的每个振幅的平均频率. </li>
	<li> 使用 OH7547 和 #34 中的 FFTs 重复步骤 6.3; 滚轮和 #34; 蠕虫. </li>
</ol> 7。傅立叶谱的建模 <ol> <li> 对线虫运动的二进制模型进行编程 (参见补充材料中包含的程序) 
	注意: 该模型是一个粗略的近似, 它首先显示了蜗杆运动的显著特征。模型可以被提炼, 因为结果与实际蠕虫比较。蠕虫形状是使用显微镜图像 13 近似的。
	<ol> <li> 创建二进制模型的连续帧, 以移动至少两个蠕虫周期 ( 图 2a )。查看补充材料中的视频;C 蠕虫视频 (CWorm) 和 W 蠕虫视频 (WWorm. avi). </li>
	</ol> </li> <li> 生成连续的衍射图案。请参阅补充材料中的视频。C 蠕虫衍射视频 (CWormDiff. avi) 和 W 蠕虫视频 (WWormDiff. avi)。
	<ol> <li> 傅立叶变换蠕虫图像的每个二进制帧。围绕蠕虫的帧的填充越大, 衍射图像的分辨率就越高. 
		注: 每个傅里叶变换帧的绝对值与相应的衍射图样成正比 ( 图 2b ). </li>
		<li> 通过将衍射图样的强度映射到对数刻度来调整衍射图案的对比度. 
		注意: 相机和眼睛的功能在一个非线性的规模。对数刻度可以模拟人们眼中的衍射图样. </li>
	</ol> </li> <li> 提取模型化的衍射信号。
	<ol> <li> 选取与光电二极管在衍射图样中的位置对应的偏心位置. </li>
		<li> 在光电二极管的位置周围添加相邻的矩阵元素以模拟光电二极管的大小。光电二极管的大小通常是0.1% 的模拟衍射模式. </li>
		<li> 记录和绘制衍射信号的大小序列。检查信号的物理合理性 ( 即 , 在周期性颠簸的情况下, 光电二极管发出的信号也应该是周期性的). </li>
	</ol> </li> <li> 傅立叶变换在7.3 获得的衍射信号, 并与实验数据进行比较. </li>
	<li> 重复不同的蠕虫病毒并进行比较. </li>
</ol>

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作者没有什么可透露的。

包括不活动的数据的延伸将扭曲结果, 因为人工较低的频率将被平均纳入结果。在原始数据中, 可以通过平坦峰值或 "切断" 峰值来识别光电二极管的饱和。按峰值强度划分每个原始数据将有助于计算激光强度的波动。
峰值频率是总颠簸频率的指示器;然而, 复杂的运动会引起衍射图样中跳动频率的干扰, 需要仔细检查。
该方法可用于研究其他线虫的运动。可以将环境更改为其他媒体。波长也可能改变。工作在可见光范围内的电磁波谱是最简单和最安全的。
更精确的模型将在未来更真实地模拟衍射光谱。未来的模型可能包括可以改变方向的蠕虫, 这不会影响频率位置, 而是相对峰值高度。一个更现实的模型将允许概率分布的颠簸频率, 这将扩大峰值的实验数据。频率的传播会考虑到颠簸频率的变化。
当前的蠕虫形状是粗的, 特别是在头部和尾部区域, 这应该比目前的模型更锥形。对信号的时间序列进行详细的分析可能会很有趣, 因为它可以给出不同突变体的运动复杂性的线索。
将该技术推广到同时对多线虫进行定性是值得考虑的。这种方法应该被理解为使用传统显微镜的现有方法的一种补充方法。这种方法在数据获取过程中不需要显微镜, 因此蠕虫可能会移出焦平面。平均频率谱在蜗杆运动中显示出明显的差异, 并可通过流行的频率峰值进行量化, 这是一种新的定量的蠕虫运动方法。衍射特征的数据分析正在进一步发展, 并有望导致多个突变体和个体的自动识别过程。

该方法比较了两种不同运动模式的菌株的实验和模拟的频率谱. 结果表明, 当线虫在水柱中游动时, 频谱依赖于时间变化, 因此不需要清晰的显微图像进行分析。此方法允许定量 real-time 分析, 并提供与传统显微镜获得的图像/视频的补充信息。弗劳恩霍夫衍射, 也称为远场衍射, 为获得活衍射数据提供了依据1,2。在衍射图样的任何一个点上的光强度是从线虫的轮廓中的每一点的叠加光的结果3。因此, 随着时间的推移收集的光强度携带的信息关于线虫的运动。分析了随时间推移的衍射信号可以识别出相应突变体的特征运动, 因为分析了运动中涉及的所有频率, 补充了传统的视频分析。在这种情况下, 通过比较两种不同菌株的频率谱, 证实了 "滚子" 与野生型线虫的运动之间的特征差异。
一些以前的特征已被证实使用频率分析的衍射信号, 如游泳频率2,4。更重要的是, 这种方法可以作为传统显微镜的一种补充方法, 在收集数据的同时, 在计算机屏幕上实时观察运动。通过考虑衍射信号的傅里叶变换信号, 可以对具有不同运动模式的蠕虫的频谱进行量化。
Fourier-based 衍射在这项工作中的多学科性质涉及生物学和物理领域。在生物的晶体结构的研究中, 长期使用的衍射在生物学5和其他领域。然而, 在这个实验中, 采样6,7创建了远场衍射模式, 使生物体以激光束为中心。采样通常用于与重建原始对象的图像的相位检索算法相结合的无透镜成像8 。当散射像线虫那样存在时, 相位检索很难实现。时间衍射特征足以评估蜗杆运动的关键频率。这种方法是较少的计算征税和提供一种光学的方式来量化的运动。这项技术可以很容易地被改编为分析突变或环境条件, 改变行为。

<table><tbody><tr><td>Tunable Helium-Neon laser</td><td>Research Electro-Optics</td><td>30602</td><td>Four wavelengths can be selected between 543 nm and 633 nm.</td></tr><tr><td>2 Front Surface Aluminum Mirrors</td><td>Thorlabs</td><td>PF10-03-F01</td><td></td></tr><tr><td>Photodiode: SI Amplified Detector</td><td>Thorlabs</td><td>PDA 100A</td><td></td></tr><tr><td>Quartz Cuvette</td><td>Starna Cells</td><td>21/G/5</td><td>Plastic cells may be used as well.</td></tr><tr><td>MatLab (Software)</td><td>MathWorks</td><td>R2016b (9.1.0.441655)</td><td>Use the fft command to simulate diffraction</td></tr><tr><td>Excel</td><td>Microsoft</td><td>14.7.1</td><td>Used for data analysis of Figure 4</td></tr><tr><td>&lt;em&gt;Caenorhabditis elegans&lt;/em&gt; Roller</td><td>&lt;em&gt;University of Minnesota Caenorhabditis elegans Center (CGC)&lt;/em&gt;</td><td>Strain: OH7547&lt;br/&gt; Genotype: otIs199.</td><td>https://cbs.umn.edu/cgc/home</td></tr><tr><td>&lt;em&gt;Caenorhabditis elegans&lt;/em&gt; Wild Type</td><td>&lt;em&gt;University of Minnesota Caenorhabditis elegans Center (CGC)&lt;/em&gt;</td><td>Strain:N2 Genotype: C. elegans wild isolate</td><td>https://cbs.umn.edu/cgc/home</td></tr></tbody></table>

fourier-based diffraction analysis of live caenorhabditis elegans

这篇手稿描述了如何使用世俗远场衍射特征对线虫进行分类。单个C. 线虫悬浮在光学试管内的水柱中。632 nm 连续波氖激光器是通过试管使用前表面反射镜。在光线穿过试管后, 至少有20-30 厘米的距离可以确保一个有用的远场 (弗劳恩霍夫) 衍射图样。当线虫在激光束内游动时, 衍射模式会发生实时变化。光电二极管被放置在衍射图样的偏心位置。光电二极管的电压信号是实时观测的, 并使用数字示波器记录。此过程重复139野生类型和 108 "滚子" C. 线虫。野生型蠕虫在溶液中表现出快速振荡模式。"滚子" 蠕虫有一个突变的角质层的关键组成部分, 干扰平滑的运动。不含饱和度和不活动的时间间隔将被丢弃。将每个平均值除以其最大值以比较相对强度是可行的。每个蠕虫的信号是傅里叶变换, 使每个蠕虫的频率模式出现。每种类型的蠕虫的信号都是平均的。对野生类型和 "滚子"线虫的平均傅里叶谱进行了明显的区别, 揭示了两种不同的蠕虫病毒的动态蠕虫形态可以用傅立叶分析来区分。每个蠕虫应变的傅里叶谱匹配一个近似模型使用两个不同的二进制蠕虫形状对应于运动时刻。实际和模拟蠕虫的平均频率分布的包络确认模型与数据匹配。这种方法可以作为许多微观物种的傅里叶分析的基线, 因为每个微生物都有其独特的傅里叶谱。

在图 1中显示的光学实验装置允许对微生物进行研究, 而不被绑在一个焦平面上。在采集数据的同时, 可以在计算机屏幕上实时观察到光电二极管的抖动信号。不寻常的模式将立即可见, 而不必详细分析视频。
模型化顺序蠕虫移动和相应的衍射模式的示例如图 2所示。模型化的衍射模式定性地类似于实验模式1 , 是模拟成功地对线虫进行建模的初步迹象。
本文所研究的两种类型的C. 线虫的时间衍射样本在图 3中显示。从定性上看, 每种线虫的以率和振幅不同。某些差异可以通过曲线拟合进行量化, 如以前的出版物1所做的那样。然而, 离散傅立叶变换揭示了有关嵌入频率的更多细节:
<img alt="Equation 1" src="/files/ftp_upload/56154/56154eq1.jpg">, (1)
其中, Fk 是数字傅立叶变换 (FT) 和Fn 是与离散时间变量n和离散频率变量 n 的原始衍射信号, 其总数为数据点。平均数字傅立叶变换允许线虫被确定由它的衍射频谱的振幅 (图 4)。野生型谱的控制频率比滚筒运动谱低。
接近野生类型的模型与滚子C. 线虫注意, 野生类型倾向于在波 (W 或 S 形状) 运动中进行捶打 (图 2a), 而滚轮倾向于偏向于一个大致类似于振荡 C 形状的一侧 (图 5)。这为不同的光谱提供了一些解释。滚子将主要形成一个 c 到一侧, 而 W 振荡可以被认为是两个相反的 c 运动。因此, W 运动比 C 运动更复杂地揭示出更多的次低频频率。这一结果在计算模型中得到证实。W 形状的频率密度比 C 形状高得多 (图 6)。这在图 4的 FFT 中得到确认, 在这里, 滚子频率更集中, 而不是完全离散。轧辊的统计数据是倾斜的, 因为滚轮可以暂时恢复为野生类型的运动。
滚子类型的平滑功率谱c. 线虫显示了一个宽峰值在 ~ 1.5 赫兹, 而游泳狂放的类型c. 线虫陈列多式联运光谱 (包括峰顶在 ~ 1.0 hz 和 1.75 hz)。光电二极管 (PD) 有一个有限的大小扩散在几个矩阵元素。由于构造性和破坏性干扰不同, 衍射图样上的单个矩阵元素或点的强度各不相同;然而, 强度变化的频率对于所有矩阵元素都是相同的, 如图 7所示。考虑到时间导数 Eq 1, 可以看出频率波动不依赖于相位矩阵, 而只取决于原始对象的波动:
<img alt="Equation 2" src="/files/ftp_upload/56154/56154eq2.jpg">, (2)
当 PD 在多个矩阵元素上传播时, 峰值位置平均到一致的频率剖面。一些变化可以预期, 可以提供线索的方向蠕虫。当蠕虫的步态改变时, 频率分布会发生变化。目前的模型是一个简单的模型, 只允许评估峰值位置, 而不是相对峰值高度。不同的运动模式将平均到不同的峰值位置。

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56154/56154fig1.jpg"> 
图1。实验装置.低功率激光束通过中性密度滤光片, 通过反射镜 M1 向下穿过试管, 将蜗杆载入镜面 M2, 并向光电二极管方向移动。<a href="//ecsource.jove.com/files/ftp_upload/56154/56154fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56154/56154fig2.jpg"> 
图2。顺序蠕虫形状和相应的衍射图案.(a) 一些选定的 W 型线虫的序列二进制图像和 (b) 相应的连续衍射模式。<a href="//ecsource.jove.com/files/ftp_upload/56154/56154fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56154/56154fig3.jpg"> 
图3。实验样品衍射特征.为 (a) OH7547 "滚子" 和 (b) N2 的衍射特征码在衍射图样中使用单光电二极管 .<a href="//ecsource.jove.com/files/ftp_upload/56154/56154fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56154/56154fig4.jpg"> 
图4。实验平均功率谱的轧辊和野生型霍夫琅衍射系列.该谱显示了在光电二极管记录的时间序列的平均傅里叶变换中存在的频率。标准偏差的高斯滤波器0.075 赫兹, 被截断在3标准偏差, 用于平滑。请注意平滑滚子频谱中的宽光谱峰值 (1.5 赫兹), 与多式平滑的野生类型频谱 (包括在 ~ 1.0 和1处的峰值) 比较75赫兹)。<a href="//ecsource.jove.com/files/ftp_upload/56154/56154fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56154/56154fig5.jpg"> 
图5。衍射形成例证.衍射模式可以建模的每一个线段的思维无穷小直线 (左)。叠加这些线 (右) 演示了由 C 形线虫产生的远场衍射图样的构造。<a href="//ecsource.jove.com/files/ftp_upload/56154/56154fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56154/56154fig6.jpg"> 
图6。滚子和野型霍夫弗衍射系列的模拟功率谱.(a) C 形状和 (b) W 形状的蠕虫与光电二极管中心在矩阵元素 200 (垂直) 和 175 (水平)。W 形状显示更高的频率密度由于更复杂的运动。<a href="//ecsource.jove.com/files/ftp_upload/56154/56154fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56154/56154fig7.jpg"> 
图7。不同光电二极管位置的滚子和野型霍夫弗衍射级数的模拟功率谱.(a) W 形状蠕虫和 (b) C 形状的蠕虫, 用于在不同位置模拟光电二极管的不同位置的单个矩阵元素。不同地点的高峰高度各不相同;但是, 特定形状的峰值位置仍然保持不变。<a href="//ecsource.jove.com/files/ftp_upload/56154/56154fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans at JoVE.com

Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans

这篇手稿描述了如何区分不同的线虫使用远场衍射签名。我们比较了139种野生类型和 108 "滚子" C. 线虫的运动, 通过平均频率与瞬时霍夫费衍射签名在一个位置使用连续波激光。

活体线虫线虫的傅里叶衍射分析

This manuscript describes how to classify nematodes using temporal far-field diffraction signatures. A single C. elegans is suspended in a water column ...

fourier-based-diffraction-analysis-of-live-caenorhabditis-elegans

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Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans

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7.9K 次观看.  Vassar College. 这篇手稿描述了如何区分不同的线虫使用远场衍射签名。我们比较了139种野生类型和 108 "滚子" C. 线虫的运动, 通过平均频率与瞬时霍夫费衍射签名在一个位置使用连续波激光。

视频: Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

This study demonstrates an inexpensive and straightforward technique that allows the measurement of physical properties such as position, velocity, acceleration and forces involved in the locomotory behavior of nematodes suspended in a column of water in response to single wavelengths of light. We demonstrate how to evaluate the locomotion of a microscopic organism using Single Wavelength Shadow Imaging (SWSI) using two different examples.
The first example is a systematic and statistically viable study of the average descent of C. elegans in a column of water. For this study, we used living and dead wildtype C. elegans. When we compared the velocity and direction of nematode active movement with the passive descent of dead worms within the gravitational field, this study showed no difference in descent-times. The average descent was 1.5 mm/sec &#177; 0.1 mm/sec for both the live and dead worms using 633 nm coherent light.
The second example is a case study of select individual C. elegans changing direction during the descent in a vertical water column. Acceleration and force are analyzed in this example. This case study demonstrates the scope of other physical properties that can be evaluated using SWSI while evaluating the behavior using single wavelengths in an environment that is not accessible with traditional microscopes. Using this analysis we estimated an individual nematode is capable of thrusting with a force in excess of 28 nN.
Our findings indicate that living nematodes exert 28 nN when turning, or moving against the gravitational field. The findings further suggest that nematodes passively descend in a column of water, but can actively resist the force of gravity primarily by turning direction.

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

The technique presented here measures the path of freely swimming microscopic species using single wavelength exposure. C. elegans are used to demonstrate shadow imaging as an inexpensive alternative to costly microscopes. This technique can be adapted to accommodate various orientations, environments and species to measure direction, speed, acceleration and forces.

单波长阴影成像<em&gt;秀丽隐杆线虫</em&gt;步态包括武力在内的估算

This study demonstrates an inexpensive and straightforward technique that allows the measurement of physical properties such as position, velocity, ...

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工程学

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 ...

生物工程

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

发育生物学

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

自由行为 秀丽隐杆线虫 的钙成像，具有良好控制，非局部化振动

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

行为学

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

神经科学

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em&gt;温度。线虫</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

活体线虫线虫的傅里叶衍射分析

摘要

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单波长阴影成像

使用激光衍射自由游泳微生物的定量步态研究

基于微流体Electrotaxis的，为按需定量分析

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

自由行为秀丽隐杆线虫的钙成像，具有良好控制，非局部化振动

线粒体疾病脑膜炎基因模型中量化动物活动的实验方法比较分析

琼脂糖微室的长期钙成像

秀丽线虫纵向成像的微流控平台

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物

活体线虫线虫的傅里叶衍射分析

摘要

探索更多视频

此视频中的章节

单波长阴影成像

使用激光衍射自由游泳微生物的定量步态研究

基于微流体Electrotaxis的，为按需定量分析

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

自由行为 秀丽隐杆线虫 的钙成像，具有良好控制，非局部化振动

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

琼脂糖微室的长期钙成像

秀丽线虫纵向成像的微流控平台

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物

自由行为秀丽隐杆线虫的钙成像，具有良好控制，非局部化振动

线粒体疾病脑膜炎基因模型中量化动物活动的实验方法比较分析