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>

<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 Fig. 4</td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans Roller</td>
			<td>University of Minnesota Caenorhabditis elegans Center (CGC)</td>
			<td>Strain: OH7547 
			Genotype: otIs199.</td>
			<td>https://cbs.umn.edu/cgc/home</td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans Wild Type</td>
			<td>University of Minnesota Caenorhabditis elegans Center (CGC)</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

Fourier Series

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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

Analysis of Contact Interfaces for Single GaN Nanowire Devices

Single GaN nanowire (NW) devices fabricated on SiO2 can exhibit a strong degradation after annealing due to the occurrence of void formation at the contact/SiO2 interface. This void formation can cause cracking and delamination of the metal film, which can increase the resistance or lead to a complete failure of the NW device. In order to address issues associated with void formation, a technique was developed that removes Ni/Au contact metal films from the substrates to allow for the examination and characterization of the contact/substrate and contact/NW interfaces of single GaN NW devices. This procedure determines the degree of adhesion of the contact films to the substrate and NWs and allows for the characterization of the morphology and composition of the contact interface with the substrate and nanowires. This technique is also useful for assessing the amount of residual contamination that remains from the NW suspension and from photolithographic processes on the NW-SiO2 surface prior to metal deposition. The detailed steps of this procedure are presented for the removal of annealed Ni/Au contacts to Mg-doped GaN NWs on a SiO2 substrate.

A technique was developed that removes Ni/Au contact metal films from their substrate to allow for the examination and characterization of the contact/substrate and contact/NW interfaces of single GaN nanowire devices.

单氮化镓纳米线设备分析接触界面

Single GaN nanowire (NW) devices fabricated on SiO2 can exhibit  a strong degradation after annealing due to the  occurrence of void formation at the ...

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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, ...

In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries

Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric vehicles.1,2 However, many challenges, such as energy density and battery lifetimes, need to be overcome before this particular battery technology can be widely implemented in such applications.3 This research is challenging, and we outline a method to address these challenges using in situ NPD to probe the crystal structure of electrodes undergoing electrochemical cycling (charge/discharge) in a battery. NPD data help determine the underlying structural mechanism responsible for a range of electrode properties, and this information can direct the development of better electrodes and batteries.
We briefly review six types of battery designs custom-made for NPD experiments and detail the method to construct the &#8216;roll-over&#8217; cell that we have successfully used on the high-intensity NPD instrument, WOMBAT, at the Australian Nuclear Science and Technology Organisation (ANSTO). The design considerations and materials used for cell construction are discussed in conjunction with aspects of the actual in situ NPD experiment and initial directions are presented on how to analyze such complex in situ data.

In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries

We describe the design and construction of an electrochemical cell for the examination of electrode materials using in situ neutron powder diffraction (NPD). We briefly comment on alternate in situ NPD cell designs and discuss methods for the analysis of the corresponding in situ NPD data produced using this cell.

原位中子衍射用特制的锂离子电池

Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric ...

Quantification of Hydrogen Concentrations in Surface and Interface Layers and Bulk Materials through Depth Profiling with Nuclear Reaction Analysis

Nuclear reaction analysis (NRA) via the resonant 1H(15N,&#945;&#947;)12C reaction is a highly effective method of depth profiling that quantitatively and non-destructively reveals the hydrogen density distribution at surfaces, at interfaces, and in the volume of solid materials with high depth resolution. The technique applies a 15N ion beam of 6.385 MeV provided by an electrostatic accelerator and specifically detects the 1H isotope in depths up to about 2 &#956;m from the target surface. Surface H coverages are measured with a sensitivity in the order of ~1013 cm-2 (~1% of a typical atomic monolayer density) and H volume concentrations with a detection limit of ~1018 cm-3 (~100 at. ppm). The near-surface depth resolution is 2-5 nm for surface-normal 15N ion incidence onto the target and can be enhanced to values below 1 nm for very flat targets by adopting a surface-grazing incidence geometry. The method is versatile and readily applied to any high vacuum compatible homogeneous material with a smooth surface (no pores). Electrically conductive targets usually tolerate the ion beam irradiation with negligible degradation. Hydrogen quantitation and correct depth analysis require knowledge of the elementary composition (besides hydrogen) and mass density of the target material. Especially in combination with ultra-high vacuum methods for in-situ target preparation and characterization, 1H(15N,&#945;&#947;)12C NRA is ideally suited for hydrogen analysis at atomically controlled surfaces and nanostructured interfaces. We exemplarily demonstrate here the application of 15N NRA at the MALT Tandem accelerator facility of the University of Tokyo to (1) quantitatively measure the surface coverage and the bulk concentration of hydrogen in the near-surface region of a H2 exposed Pd(110) single crystal, and (2) to determine the depth location and layer density of hydrogen near the interfaces of thin SiO2 films on Si(100).

We illustrate the application of 1H(15N,&#945;&#947;)12C resonant nuclear reaction analysis (NRA) to quantitatively evaluate the density of hydrogen atoms on the surface, in the volume, and at an interfacial layer of solid materials. The near-surface hydrogen depth profiling of a Pd(110) single crystal and of SiO2/Si(100) stacks is described.

通过与核反应分析深度剖面的表面和界面层和颗粒材料氢含量的定量

Nuclear reaction analysis (NRA) via the resonant 1H(15N,&#945;&#947;)12C reaction is a highly effective method of depth profiling that quantitatively and ...

Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages

Synchrotron radiation micro-tomography (SR&#181;T) is a non-destructive three-dimensional (3D) imaging technique that offers high flux for fast data acquisition times with high spatial resolution. In the electronics industry there is serious interest in performing failure analysis on 3D microelectronic packages, many which contain multiple levels of high-density interconnections. Often in tomography there is a trade-off between image resolution and the volume of a sample that can be imaged. This inverse relationship limits the usefulness of conventional computed tomography (CT) systems since a microelectronic package is often large in cross sectional area 100-3,600 mm2, but has important features on the micron scale. The micro-tomography beamline at the Advanced Light Source (ALS), in Berkeley, CA USA, has a setup which is adaptable and can be tailored to a sample&#39;s properties, i.e., density, thickness, etc., with a maximum allowable cross-section of 36 x 36 mm. This setup also has the option of being either monochromatic in the energy range ~7-43 keV or operating with maximum flux in white light mode using a polychromatic beam. Presented here are details of the experimental steps taken to image an entire 16 x 16 mm system within a package, in order to obtain 3D images of the system with a spatial resolution of 8.7 &#181;m all within a scan time of less than 3 min. Also shown are results from packages scanned in different orientations and a sectioned package for higher resolution imaging. In contrast a conventional CT system would take hours to record data with potentially poorer resolution. Indeed, the ratio of field-of-view to throughput time is much higher when using the synchrotron radiation tomography setup. The description below of the experimental setup can be implemented and adapted for use with many other multi-materials.

For this study synchrotron radiation micro-tomography, a non-destructive three-dimensional imaging technique, is employed to investigate an entire microelectronic package with a cross-sectional area of 16 x 16 mm. Due to the synchrotron&#39;s high flux and brightness the sample was imaged in just 3 min&#160;with an 8.7 &#181;m spatial resolution.

利用同步辐射微断层调查的多尺度三维微电子封装

Synchrotron radiation micro-tomography (SR&#181;T) is a non-destructive three-dimensional (3D) imaging technique that offers high flux for fast data ...

Measuring Spray Droplet Size from Agricultural Nozzles Using Laser Diffraction

When making an application of any crop protection material such as an herbicide or pesticide, the applicator uses a variety of skills and information to make an application so that the material reaches the target site (i.e., plant). Information critical in this process is the droplet size that a particular spray nozzle, spray pressure, and spray solution combination generates, as droplet size greatly influences product efficacy and how the spray moves through the environment. Researchers and product manufacturers commonly use laser diffraction equipment to measure the spray droplet size in laboratory wind tunnels. The work presented here describes methods used in making spray droplet size measurements with laser diffraction equipment for both ground and aerial application scenarios that can be used to ensure inter- and intra-laboratory precision while minimizing sampling bias associated with laser diffraction systems. Maintaining critical measurement distances and concurrent airflow throughout the testing process is key to this precision. Real time data quality analysis is also critical to preventing excess variation in the data or extraneous inclusion of erroneous data. Some limitations of this method include atypical spray nozzles, spray solutions or application conditions that result in spray streams that do not fully atomize within the measurement distances discussed. Successful adaption of this method can provide a highly efficient method for evaluation of the performance of agrochemical spray application nozzles under a variety of operational settings. Also discussed are potential experimental design considerations that can be included to enhance functionality of the data collected.

We present protocols to be used in the measurement of spray droplet size from agricultural nozzles used in both aerial and ground based agrochemical applications. These methods presented were developed to provide consistent and repeatable droplet size data both inter- and intra-laboratory, when using laser diffraction systems.

从农业喷嘴采用激光衍射测量喷雾的雾滴尺寸

When making an application of any crop protection material such as an herbicide or pesticide, the applicator uses a variety of skills and information to ...

Characterization of Ultra-fine Grained and Nanocrystalline Materials Using Transmission Kikuchi Diffraction

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and nanocrystalline materials. The traditional techniques associated with scanning electron microscopy (SEM), such as electron backscatter diffraction (EBSD), do not possess the required spatial resolution due to the large interaction volume between the electrons from the beam and the atoms of the material. Transmission electron microscopy (TEM) has the required spatial resolution. However, due to a lack of automation in the analysis system, the rate of data acquisition is slow which limits the area of the specimen that can be characterized. This paper presents a new characterization technique, Transmission Kikuchi Diffraction (TKD), which enables the analysis of the microstructure of UFG and nanocrystalline materials using an SEM equipped with a standard EBSD system. The spatial resolution of this technique can reach 2 nm. This technique can be applied to a large range of materials that would be difficult to analyze using traditional EBSD. After presenting the experimental set up and describing the different steps necessary to realize a TKD analysis, examples of its use on metal alloys and minerals are shown to illustrate the resolution of the technique and its flexibility in term of material to be characterized.

This paper provides a detailed method to characterize the microstructure of ultra-fine grained and nanocrystalline materials using a scanning electron microscope equipped with a standard electron backscatter diffraction system. Metal alloys and minerals presenting refined microstructures are analyzed using this technique, showing the diversity of its possible applications.

超细晶和纳米晶材料的表征采用透射菊池衍射

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and ...

Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing

Probing remote matter with laser light is a ubiquitous technique used in circumstances as diverse as laser-induced breakdown spectroscopy and barcode scanners. In classical optics, the intensity that can be brought to bear on a remote target is limited by the spot size of the laser at the distance of the target. This spot size has a lower bound determined by the diffraction limit of classical optics. However, amplified femtosecond laser pulses generate intensity sufficient to modify the refractive index of the ambient air and undergo self-focusing. This self-focusing effect leads to the generation of highly intense laser filaments which maintain their intensity and small sub-millimeter diameter size at distances well beyond the classical Rayleigh length. Such intensity provides the capability of remote scanning, imaging, sensing, and spectroscopy with enhanced spatial resolution. We describe a technique for generating filaments with a femtosecond regenerative chirped-pulse amplifier, and for using the resulting filament to conduct imaging and spectroscopic measurements at remote distances of at least several meters.

High-intensity femtosecond pulses of laser light can undergo cycles of Kerr self-focusing and plasma defocusing, propagating an intense sub-millimeter-diameter beam over long distances. We describe a technique for generating and using these filaments to perform remote imaging and sensing beyond the classical diffraction limits of linear optics.

飞秒激光丝在亚衍射有限成像和遥感中的应用

Probing remote matter with laser light is a ubiquitous technique used in circumstances as diverse as laser-induced breakdown spectroscopy and barcode ...

Quantitative Analysis by Thermogravimetry-Mass Spectrum Analysis for Reactions with Evolved Gases

During energy conversion, material production, and metallurgy processes, reactions often have the features of unsteadiness, multistep, and multi-intermediates. Thermogravimetry-mass spectrum (TG-MS) is seen as a powerful tool to study reaction features. However, reaction details and reaction mechanics have not been effectively obtained directly from the ion current of TG-MS. Here, we provide a method of an equivalent characteristic spectrum analysis (ECSA) for analyzing the mass spectrum and giving the mass flow rate of reaction gases as precise as possible. The ECSA can effectively separate overlapping ion peaks and then eliminate the mass discrimination and temperature-dependent effect. Two example experiments are presented: (1) the decomposition of CaCO3 with evolved gas of CO2 and the decomposition of hydromagnesite with evolved gas of CO2 and H2O, to evaluate the ECSA on single-component system measurement and (2) the thermal pyrolysis of Zhundong coal with evolved gases of inorganic gases CO, H2, and CO2, and organic gases C2H4, C2H6, C3H8, C6H14, etc., to evaluate the ECSA on multi-component system measurement. Based on the successful calibration of the characteristic spectrum and relative sensitivity of specific gas and the ECSA on mass spectrum, we demonstrate that the ECSA accurately gives the mass flow rates of each evolved gas, including organic or inorganic gases, for not only single but multi-component reactions, which cannot be implemented by the traditional measurements.

Precise determination of the evolved gases&#39; flow rate is key to study the details of reactions. We provide a novel quantitative analysis method of equivalent characteristic spectrum analysis for thermogravimetry-mass spectrum analysis by establishing the calibration system of the characteristic spectrum and relative sensitivity, for obtaining the flow rate.

热重分析-质谱分析对演化气体反应的定量分析

During energy conversion, material production, and metallurgy processes, reactions often have the features of unsteadiness, multistep, and ...

Crack Monitoring in Resonance Fatigue Testing of Welded Specimens Using Digital Image Correlation

A procedure using digital image correlation (DIC) to detect cracks on welded specimens during fatigue tests on resonance testing machines is presented. It is intended as a practical and reproducible procedure to identify macroscopic cracks at an early stage and monitor crack propagation during fatigue tests. It consists of strain field measurements at the weld using DIC. Images are taken at fixed load cycle intervals. Cracks become visible in the computed strain field as elevated strains. This way, the whole width of a small-scale specimen can be monitored to detect where and when a crack initiates. Subsequently, it is possible to monitor the development of the crack length. Because the resulting images are saved, the results are verifiable and comparable. The procedure is limited to cracks initiating at the surface and is intended for fatigue tests under laboratory conditions. By visualizing the crack, the presented procedure allows direct observation of macrocracks from their formation until rupture of the specimen.

Digital image correlation is used in fatigue tests on a resonance testing machine to detect macroscopic cracks and monitor crack propagation in welded specimens. Cracks on the specimen surface become visible as increased strains.

使用数字图像相关性对焊接试样共振疲劳测试中的裂纹监测

A procedure using digital image correlation (DIC) to detect cracks on welded specimens during fatigue tests on resonance testing machines is presented. It ...