We acknowledge Dr. Dietmar Schwahn (Forschungszentrum J&#252;lich GmbH) for support and stimulating discussions regarding the upgrades performed on the KWS-2 in 2010-2015. The constant help from the Central Institute of Engineering, Electronics, and Analytics (ZEA) and the JCNS-1 (Neutron Scattering) and JCNS-2 (Scattering Methods) Institutes in Forschungszentrum J&#252;lich GmbH during the design, installation, and commission of components, devices, and control software for the new working modes of the KWS-2 is gratefully acknowledged. We are thankful to Matthew Binns and Christopher J. Van Leeuwen (both at Louisiana State University) for the professional editing of this manuscript.

1.装入样品池<ol><li>分散具有不同尺寸（R = 150，350，500，1000的半径，和4000埃）和σR的大小多分散性的聚苯乙烯球形颗粒<img alt="公式5" src="/files/ftp_upload/54639/54639eq5.jpg" />在水溶液8％以1％的体积分数（90％D 2 O和10％H 2 O的混合物）。 </li><li>转移聚苯乙烯颗粒的六大解决方案，D 2 O / H 2 0，的C 28在D 2 O中的溶液H 57 -PEO5，和D 2 O / H 2 O和D 2 O溶剂石英细胞（ 图3 ）使用巴斯德吸管。填充每个石英细胞到颈部。关闭石英细胞与他们的瓶塞。 注意:用样品填充所述石英细胞的操作必须在FRMⅡ的样品制备的实验室通过与其中定义的特殊工作条件符合来进行。 </li><li>的InstaLL填充石英细胞进入样品架的铝筒（ 图3）。将每个填充石英池在盒的空腔，并检查石英小区是否通过确定所述样品是否完全覆盖在于提供了一种用于中子暗盒窗口开口填充有足够的样本。放置在盒上其他位置的校正和标准样品（空石英电池中，硼碳化物板，和有机玻璃板），并留下自由的空光束测量一个位置。 </li><li>包括与使用螺栓（M3x5）镉涂层铝盖板（ 图3）墨盒。通过使用特殊的铝螺钉（ 图4）固定样品架中的Al-框架上的墨盒。 </li></ol> 2.定位样品台的样品架/样品环境<ol><li>通过调整确定在样品架的样本位置的必要的空间准直鼻子的长度（ 图5）。选择用于从准直机头的控制系统所存储的配置中的样本保持器的适当的配置。 </li><li>在使用内六角螺钉，M6x40（ 图6）的光学平台的预定位置安装在样品台上的相应样本保持器/样品的环境。 </li><li>通过使用在其外侧的旋钮，保持控制旋钮激活直到门到达端位置，这将是一个发光信号指示关闭机动滑动引线门（ 图5）。 注意:如果没有门完全关闭和最终开关激活时，光束快门不能打开，手动或从测量控制软件;测量软件将需要针对此问题额外的检查开始测量之前。 </li></ol> 3.规划实验<ol><li>选择adequ吃实验结构和模式，用于执行在问答范围的调查是适合的结构和通过样品揭示了相关效应的长度尺度。检查仪器4（ 图7和表2）的动态范围。 </li><li>选择用于实现对通过知道表征样品进行调查8的近似大小，浓度和对比度因子基于所述散射强度的估计可与样品适当强度的充分的实验配置和模式。这样做是为了优化测量时间为目标测量统计并在样本的情况下，样品的稳定性具有短的稳定时间匹配完成。检查不同的实验配置4（ 补充图1和表2）的强度图。 </li><li>选择适当的实验配置和模式用于基于多分散性的大小程度和试样8中的散射的对象的浓度的近似知识妥善解决。这是为了使所发生由于订货样品中的效果优良的散射特性的分辨率完成。检查调整波长分辨率5（ 表2）的可能性。 </li></ol> 4.准备测量软件和进行和可视化实验<ol><li>通过以激活主菜单（ 补充 图3）中的KWS-2仪器的测量控制计算机终端窗口键入KWS2TC开始测量软件。使用左边的功能集来定义基本电机位置（配置），选择样本和设置条件（定义），开始测量和监督所有电机（控制）和监督权的实际探测器（实时显示）。 </li><li>为了激活kws2-配置菜单（ 补充图4）来定义用户数据，并配置为在样品上的装置和字段的基本马达位置和设定点选择主菜单中的配置的功能。 <ol><li>选择的UserData功能（ 补充图4），满山遍野用户名，电子邮件，文件名的第一部分，并在用户数据菜单测量评论（查看/编辑）。通过单击保存退出菜单。 注意:不要使用特殊字符，如@，$，％ 等 ，文件名前缀。避免在整个实验过程中使用特殊字符。 </li><li>为了激活示例配置菜单（ 图补充4）选择采样功能（ 补充图4）。满山遍野样品名称，样品束窗口 - 尺寸，试样厚度和注释每个SAMPLe和从菜单的左垂直列表中选择的位置。在完成定义后保存每个样品的配置。通过单击关闭退出菜单。 </li><li>保存在配置菜单上的功能集的文件功能下的所有配置。 </li></ol></li><li>为了激活kws2定义菜单（ 补充图5）来定义的实验装置和测量程序选择主菜单（ 补充图3）定义的功能。 <ol><li>为了激活选择样本菜单（ 补充图5）选择采样功能。 <ol><li>选择十二个样品必须进行测量（ 见图3）从已知样本的列表在左侧垂直场，并使用蓝色箭头它们移动到选定的样品字段。通过使用蓝色垂直箭头订购选择的样本的列表。 </li><li>检查样品Parameters和，调整名称，厚度和评论，如果必要的。通过单击保存或关闭退出菜单。 </li></ol></li><li>选择检测功能，以激活测量菜单（ 补充图6）的定义。 <ol><li>通过在测量面积选择标准选择静态测量类型。在结束条件区域，选择正确的时间单位现场测量时间。 </li><li>在选择探测器和准直距离区，由波长（选择字段），检测距离（检测距离场），数据采集模式（TOF场），旨在波长分辨率选择合适的值选择实验设置和工作模式（DLambda /λ场），测量时间（时间字段），镜头配置（镜头场），和准直距离（距离瞄准场）。 </li><li>单击新建按钮后一种配置是为了修复它，并把它存储在LO完全定义WER表。定义下一个配置并存储在一个类似的方式，直到整组的配置的（ 补充图6）被完成。点击保存或测量菜单的定义关闭时，实验设置和工作模式的调整结束。 </li><li>根据三个环（排序条件）在由该程序（ 补充图7）生成的菜单的底部示出排序的测量结果列表。删除未通过标记相应的行，然后点击"X"按钮标记为红色所需的测量。通过检查场中央的总定义的测量时间，标记为红色，在kws2定义菜单，始终保持活跃在屏幕上，需要调整测量时间每次测量。 </li><li>离开菜单，保存或关闭并返回到kws2定义菜单（ 补充图5）。关闭kws2定义菜单和返回主菜单（ 补充图3）。 </li></ol></li></ol></li><li>为了激活测量控制菜单（ 图补充8）选择在KWS2测量软件（ 补充图3）的主菜单中的控制功能。 <ol><li>登录的用户名和密码，将由仪器科学家连通并锁定会话用于产生脚本，它将执行上传的测量程序的命令。为了检查上传的测量程序选择循环定义。 </li><li>按下启动按钮，并回答了约样品位置门的实际状态和梁快门程序生成的问题。测量程序将被启动。为了使正在进行测量的可视化（电机的位置和仪表元器件的状态选择当前值，计数率和进化论在时间积分强度的n个）。 注:检测器和检测器和监视器的计数率上的积分强度示出并可以用来改变测量程序，单独或该组测量值的全部。 </li></ol></li><li>允许测量根据所定义的测量程序进行，并完成。 注意:测量可通过激活停止功能并选择所需的选项（继续，有或没有保存文件以停止电流测量，或停止整个程序），当积分强度收集到一个被中断或停止一定的时间点被认为是足够的，或者当故障在测量序列被识别。 <ol><li>通过点击循环定义选项下的打印按钮生成测量会话日志（ 补充图8）时所定义的测量程序已经停止或补偿leted。 </li></ol></li><li>为了激活KWSlive_MainWindow接口（ 补充图9）选择在KWS2测量软件（ 补充图3）的主菜单实况显示。 <ol><li>在类型屏幕，选择使用GEDET或PSD以分别以可视化的主要或次要（高清晰度）探测器收集的数据。通过选择所述三维（表面）在显示模式的可视化模式中，二维（轮廓），或一维（径向平均）模式。为了使数据的相对Q上呈现为n强度（未校正）输入的情节选项（线性或对数标度），并在径向平均选项菜单的字段的参数值（波长和检测距离L D）。 </li><li>为了形象化在TOF模式（无论是主或次级检测器）收集的数据选择所需的飞行时间的信道。 </li></ol></li></ol> 5.数据分析<ol><li>通过在KWS-2仪器的数据分析计算机的终端窗口中键入qtiKWS命令启动数据处理软件。选择主界面右侧的新脚本选项。 </li><li>为了激活数据分析功能选择在主界面（ 补充图10）的上的功能菜单的DAN选项。为了激活对KWS-2测得的数据使用主探测器的数据分析模式选择右侧菜单上的选项的KWS-2仪器。定义，其中所测量的数据文件的位置和其中所述校正数据文件将被存储的文件夹。 </li><li>选择工具选择和激活右侧菜单（ 补充图11）上的头（多个）功能并生成信息表包含要处理的文件。点击左侧集绿色箭头的定义表名在页眉（S）字段。通过点击右侧设置页眉（S）领域的绿色箭头，然后选择测量文件加载测量文件。 注:包含关于每个测量文件的完整信息的信息表被存储在项目的下部探险状菜单。将在项目稍后产生的所有结果将被存储在那里。 </li><li>激活在右侧的菜单（ 补充图12）的掩码函数和生成的活性掩模限定将要考虑的用于数据处理的检测器的面积。输入限定的各向同性散射图案的分析的情况下，矩形的荫罩的左下和右上拐角中的边缘和值波束停止字段。 </li><li>激活在右侧的菜单（ 补充图13）的灵敏度函数和通过输入在标记为绿色音响产生用于某些配置的检测器灵敏度视场的标准样品（树脂玻璃），空束（EB），并阻断光束（B 4 C）的测量值的游程数。 <ol><li>点击集绿色箭头旁边的黄色字段（传输），用于计算标准样品的传输。产生并通过选择计算作为新命名灵敏度矩阵，并且通过在下部菜单使用适当的情节功能可视化生成矩阵。在其他配置的情况下，重复此过程。 </li></ol></li><li>激活在右侧的菜单（ 补充图14）的数据处理功能，并产生为了校正，校准，并执行数据的径向平均的校正和校准表和脚本表。 <ol><li>定义的，通过使用水平滑块上右侧菜单（红色箭头）的顶部在实验中使用的条件的数目。通过输入每个实验条件填充黄色铅笔指示的字段运行空细胞数（EC），阻断光束（B 4 C），和标准样品，有机玻璃（绝对校准。FS），用于标准校正（绝对校准。EB）空光束，并阻止光束为标准校正（绝对校准。B4C）。 </li><li>具有很强的前锋在中心场散射输入测量的运行数字。输入在EB字段空束的运行次数并通过检查旁边的TR（EC-至EB）功能的框选择为样本的传输的计算相应的实验条件。 </li><li>点击由一组以从定义的文件加载所需的数据处理的信息，并计算出空单元的传送旋转在垂直一系列选项（ 补充图14）的绿色箭头的指示的每个按钮。点击每个黄色塔的顶部，以定义列名。 </li><li>点击为了新建按钮，生成并命名数据文件，将B的表Ë处理。点击以加载将要处理的数据文件添加按钮。点击与该组，以便计算每种样品的透射旋转脚本表格工具区域下的绿色箭头的指示的TR按钮。检查所生成的表（ 补充图14）的结果。 </li><li>为了节省所有结果在当前qtiKWS会议（项目）表或矩阵选择项目中的接口（ 补充图14）的右下角。点击以便执行二维数据的校正和校准的I [X，Y]按钮。点击以执行校正，校准和数据的径向平均的I（Q）的按钮。使用下图（ 图补充15）选项图形功能绘制的结果。 注:所有结果将作为将被保存在步骤5.2中定义的外部文件夹外部文件的时候，在日生成E中的界面的右下角，文件选择，而不是项目。 </li></ol></li><li>在右侧的菜单（ 补充图11）选择的工具和激活TOF |为了分割收集与在飞行时间的工作模式到单个文件对应每次主检测器的数据的RT选项（ 补充图16）渠道。 <ol><li>单击TOF ::计算参数的功能和负载一个文件，从中提取有关TOF条件的信息。点击TOF | RT ::求和VS数量::了解功能和负载感兴趣的文件，在实时或TOF模式测量，以产生像上的左侧示出的总和-TOF-文件表工作界面。积的积分强度作为使用在上层的功能菜单中的图形函数下的图形选项从总和-TOF-文件时道（ 补充图16）的功能。 </li><li>定义处理P.arameters在TOF功能的领域。单击TOF | RT ::所有选定步骤::以加载将被分离成对应于各所定义的时隙单个文件中的数据文件继续按钮。 注:包含在每个时隙测量的数据文件被生成并存储在在步骤5.2中定义的文件的位置和接收原始TOF文件的名称，后面的时隙的数目。 </li><li>进行如在步骤5.6为了分析具有改进的分辨率的测量的数据，对应于Δλλ 目的 ，使用斩波器。 </li></ol></li><li>为了激活使用二次高分辨检测与KWS-2测得的数据进行数据分析模式，从主界面（ 补充图17）的右侧菜单上的选项的KWS2-HRD仪器。激活右侧菜单屏蔽功能（ 补充图18A）和发电机密封e本有源掩模限定检测器的有源区。 <ol><li>选择主界面（ 如图补充18B）的上部的功能菜单中的选项DANP。选择右侧菜单中的选项ASCII.2D。激活以定义将被认为是数据分析的检测器上的特殊扇区的2D屏蔽功能。 </li><li>介绍在现场中心束一站式中心。选择掩码矩阵和面具::条件值为0忽视的特殊面膜的外侧区域。选择角扇区和点击扇区字段的右侧的彩色按钮。与高分辨率检测器测得的，如在步骤5.5和5.6中的数据继续进行。 </li></ol></li><li>保存qtiKWS项目（除上级菜单的文件选项下的功能）。 </li></ol>

<ol>
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The authors have nothing to disclose.

软物质和生物物理系统的典型特征是跨越宽长度尺度，从纳米到微米的结构的相关性和相互关联的微观结构和形态的水平。理解的形成，而这些体系的形态和其微观特征和宏观性质之间的关系的进化的机构，在整个长度规模和相关的环境条件（ 例如，温度，压力，pH值下，探讨其显微组织是很重要的，湿度等 ）。通常情况下，小角散射中子（SAN）或同步加速器X射线（SAXS）技术涉及这样的研究。中子与同步辐射X射线的强度的缺点是通过使用比较大的Δλ/λ，然而导致仪器的分辨率的恶化得到补偿。然而，SANS由于提供了对照提供的可能性独特优势AST变型中，特别是氢同位素之间。因此，SANS是软物质和生物物理系统的研究，为此它提供了独特的结构和形态信息的专门用于实验的方法。最SANS的衍射仪上的针孔原理（ 图2A）全世界21的工作，这使得针对低Q的分辨率。实际上，所有的高通量SANS衍射仪具有1×10 8 N厘米-2秒-1量级的类似最大通量。基于宽松的波长的分辨率，KWS-2具有近一倍通量2,4。日前，非常专业的SANS衍射仪开始运作与应用，如用于调查一个特定范围内的最佳特性，以服务于非常小的散射向量22，23。与专门TOF-SANS衍射仪的稳态反应堆24或spallatio非常最近佣金n个源25，26，在给定的实验装置和有关的实验的分辨率的选择更大的灵活性和优化大规模增动态Q系列，提供。对于KWS-2 SANS衍射，通用性和对在软物质和生物物理学领域非常具体的结构研究所需的性能的一个高层次上一个否则古典SANS仪启用。优化，灵活性和自发性在设计和进行复杂的研究，由所描述的协议所支持的，是通过优化实验参数（例如，强度，长度尺度，空间分辨率和时间分辨率）和复数样本的组合来实现环境。利用在引言中列举和由图8-15给出的结果所支持的多种工作模式，该KWS-2在一个简单而实用的方式增强了经典SANS衍射的性能在一个稳定的中子源（反应堆）超出这些文书的传统界限。 这个协议提出了一个普通用户必须执行来定义，并进行简单的实验方案涉及仅在环境热力学条件（温度，压力，相对湿度）和静态条件下样品的调查（无结构形成动力学或步骤变换，没有剪切或流量）。几个温度控制持有人或特殊样品的环境（ 表1和补充图19），如压力单元，流变仪，或湿度的细胞，可用并且可以最佳地安装，并与从仪器团队特别协助调整。该协议不提供有关这种设备的设置和控制的说明。的定义和外部控制器的激活需要使用另一个更复杂的协议。该协议提出了情况下具有窄的矩形形状（ 图3）的石英样品池的工作。然而，广泛的细胞几何形状和类型（ 补充图19）的被提供给用户，以便提供增加的灵活性和效率在进行实验。在使用这种电池的情况下，本协议之后可能会有在步骤4.2.2所讨论的参数的调整。测量控制软件已经发展到提供用户在追求自己的科学目标和优化仪器的技术操作更大的灵活性。所有的调整和特殊功能和仪器的部件配置通过仪器团队完成的。在配置上，定义，和仪器的利用率科学用户的参与被简化，只特别地限定于处于与实验会议的科学问题连接的那些方面。有限公司nfiguration文件被预定义以涵盖所有特殊的实验问题，如特持有人在梁的定位，在光束的样品定位（坐标x，y，Φ，ω在样品台上，旋转工作台，或摇篮在补充图5），对不同波长的检测器和波束停止位置的调整，斩波参数的调整（频率和开口窗）为不同的波长，在检测的距离和目标的分辨率等 。此外，当前协议不描述如何实时模式可以在KWS-2一起使用。使用更复杂的协议，还需要为了进行时间分辨SANS实验。 此外，该协议提出如何测得的数据可用于从仪器和引用不同散射贡献来校正，并且为了获得样品的differenti校准人散射截面，dΣ/dΩ，表示在-1。这个量包含有关样品的整个结构和形态的信息，并在对应于一广泛的长度尺度，在其上调查系统的特征性结构的相关性和相互关联的大小的水平出现一个宽Q系列，进行测定。散射截面dΣ/dΩ因而涉及在一个角度Θ在静态散射实验测得的强度，I S = F（Θ）到样品的结构特性。 对于dΣ/dΩ的对于感兴趣的系统中的评价，除了系统的测量，需要以校正数据为任何外部散射（ 即，环境，样品池，溶剂或缓冲液中的情况下，解决方案的其他的测量溶质系统， 等 ），并在绝对单位的校正数据校正<sup<html> &gt; 8。外部背景（样品池或容器）中，参照样品（溶剂或缓冲液），样本传输（用于在绝对单位校正结果的正确背景减除和校准）时，检测器的电子背景下，检测器灵敏度，并且归一化的标准样品（在检测器的效率是固有对于面积探测器不均匀性）也应进行测量。对于KWS-2，树脂玻璃（聚甲基丙烯酸甲酯）用作标准样品。这就是所谓的二级标准并定期校准针对主标准样品，这是钒。钒提供了一个非常微弱的散射强度，需要收集相应的统计数据很长的测量时间;因此，它是不切实际的SANS目的。从兴趣I S的样品，并从标准样品I ST收集的强度可表示如下: T"&gt; <img alt="式（1）" src="/files/ftp_upload/54639/54639eq1.jpg" /> [1] <img alt="式（1）" src="/files/ftp_upload/54639/54639eq2.jpg" /> [2] 其中I 0表示传入的强度（由准直系统交付），t为厚度，A是暴露在光束的区域，T是传输，并且Δψ是在其中检测单元是从看到的立体角样品位置。如果两个样品和标准在相同条件下测量相对于所述入射光束（ 即，L C，A C和A S和λ和Δλ/λ），I 0和A是相同的，在立体角是表示为A D / L D（与表示检测单元的区域A D）。通过将两个关系，散射横secti在样品的获得为: <img alt="式（1）" src="/files/ftp_upload/54639/54639eq3.jpg" /> [3] 其中我圣表示为平均值（标准作为一个非相干散射系统提供了一个平坦的散射图案）。该I S是相对于校正在细胞（容器）样品的测量强度为空电池ⅠECell的贡献和为封闭梁，I B的检测器上的背景后获得的。吨圣 Ť 圣 （dΣ/dΩ） 圣 ，其中包含散射和标准样品的物理参数的因子，取决于中子波长λ和通常从标准样品的校准已知的。因此，在数据还原软件4列。公式中的参数和数量。 3个从校准再修改的知ES和实验装置（ 叔S，L D）的定义，形成所谓的校准系数k。出现在方程的强度和样品变速器T 秒 。 3，必须进行测量。数据分析程序qtiKWS使得校正，校准，和径向的实验数据的平均值和dΣ/dΩ为调查样品以灵活和通用的工作模式的实现。与qtiKWS软件生成的最终结果表示为具有四个列的表:Q，I，ΔI，Δσ其中I表示dΣ/dΩ和Δσ为Q分辨率5。 从实际的观点来看，与KWS-2，组合SANS和USANS调查可以进行，其优点是对样品的几何形状和热力学条件保持不变。大形态，显示MULTIPLE结构层次，从纳米跨越宽长度尺度到微米尺寸可以在一个直接方式进行研究，如如图12所示。除了在常规针孔模式中测得的散射曲线中观察到的小规模结构的限制R C中，通过激活透镜和高分辨率检测器，由该聚形成的圆筒状的核-壳微胶粒的大型结构限制L C - （亚己基-氧化物-共-环氧乙烷）在D 2 O中14 PHO10k-PEO10k二嵌段共聚物（完全质子化）可以在非常低的Q值在更多的问答 -range工作模式进行观察。圆柱形胶束的特征在于大约300埃的总厚度和大约7000埃的长度，由与芯-壳圆柱形外形9,14的实验结果的拟合所揭示。因此，某些敏感的影响，如热 - 反应凝胶或形成和晶体的生长线或部分结晶形态可与KWS-2可以清楚地探讨，不同于涉及两个或多个不同的仪器和样品的几何形状的传​​统方法。 如图11所示，相关系统和有序结构可以在一个非常灵活的方式适应的分辨率进行研究，无需花费时间和精力上的安装复杂的单色系统，这将涉及额外的护理和安全方面的问题。而且，涉及斩波器和飞行时间的数据采集模式，单分散性的软物质系统或具有低多分散性的大小配合，可以非常精确地在仍然高强度的特点5。 由于使用高度稀释的系统或不利对比度条件由弱散射产生的障碍可通过使用基于关于样本较大光束尺寸甚至更高强度来克服，同时保持解析度。 图13A从R的半径的聚苯乙烯颗粒报告散射图案= 150埃，使用的透镜和一个二次光束尺寸范围毫米10毫米之间×10，在传统的针孔模式中使用的典型大小中的高强度的模式中测得，30毫米×30毫米。此外，从与圆形梁50毫米的直径（全透镜尺寸）测定的结果被示出。平行地，在以往的针孔模式中得到的归一化的结果呈现。使用26透镜与λ= 7埃，同一入口孔径大小A C作为用于常规针孔模式（ 图2B）的中子，同时保持恒定的束大小获得强度的增益在约12倍的样品（第）的检测器，如在图13B-C中所示的。 27透镜系统具有在环境温度下为约32％的透射。冷却到50 K的温度时，透镜传输的增加是由于抑制散射对在镜片材料中声子。 26抛物线透镜系统具有用于为50mm的圆形的光束的大小，约65％的透射当光束穿过透镜的整个体积，以及约92％的10毫米×10毫米的二次光束尺寸当只有透镜材料的非常量停留在光束。与镜头的高强度模式，在通常是在一个大的探测距离遇到的是在弱对比的条件的情况下特别有问题的弱散射的情况下提供好处。另外，当样品是仅在次短时间稳定，使用这种模式的表示有明显的优势，如证明别处15。 另一方面，在生物系统中的情况下，小的样品体积通常可用于实验。在具有几纳米的尺寸生理条件小的生物分子递送DOMI上述弱散射信号从缓冲溶液恶性散射。这样的信号可以与KWS-2进行测量，从针孔模式的低分辨率设置在仪器的高强度受益，使用短准直长度L C =2μm或4米和短路检测距离L1（D）= 1米，2米，或4微米。 图14表示从β淀粉样蛋白的蛋白质中的氘化六氟异丙醇dHFIP散射图案（Aβ1-42中，M W = 4.5 kDa）的单体，施加用于从所述缓冲器中的散射信号的修正后得到。数据的模型拟合交付约16±1单体尺寸为16。对于每个实验条件数小时（检测距离L D和样品类型）一个长的测量时间被卷入，虽然测量在短路检测距离进行。旧的探测器，它已经显示出对于计数率的限制，阻碍了中使用的短距离准直的L C，因此，在器械的使用的最大通量。与新的检测系统能够使用的全部中子通量的委托，这样弱的强度将在更短的时间，并在将来改进统计测量。 最后，刺激敏感效应可以以灵活和容易的方式通过使用KWS-2的特别的辅助设备的影响。一个例子被报告于图15，其示出了在D 2 O中缓冲，并从在不同压力下收集的缓冲器从溶菌酶蛋白的SANS图案。特别注意了从溶菌酶分子的背景和前向散射的研究，按照由PSI，瑞士做了设计，内部制造新的压力电池的性能测试中使用。该结果类似于由Kohlbrecher 等获得的那些。在类似的研究以测试构造的原始压力小区模型有17，18。与KWS-2，进一步的数据已经采集自5000巴的压力已经达到。从蛋白质的前方散射强度的进化遵循线性行为，如在PSI，瑞士18在研究中观察到的。

柔软和生物材料显示了丰富多样的形态为特征的功能，如自组织和基本单元的自组装到较大的，复杂的聚集体。它们还显示有大量的自由度协同相互作用;的结构单元，以及外部场从而高灵敏度之间的弱相互作用;和时空相关性可以跨越广泛的范围内，从纳米到毫米，从纳秒到数天。因为大范围的相关长度 - 和时间表的，这些材料的性能的实验表征是非常具有挑战性的。中子散射技术起到结构，动力学，并且这样的复杂系统的热力学特性的调查中起重要作用。作为独特的探针，中子提供1 H和2 H（氘，D）的氢同位素之间不同的相互作用的优点。大不同ENCE在氢和氘之间的相干散射长度密度代表的对比度变化和对比匹配方法的基础。由于大部分的软物质和生物系统包括烃，氢气/氘（H / D）的取代提供了在宽的范围内变化的化合物的相干散射长度密度的可能性。利用这种技术，在一个复杂的系统选定成分可由同位素交换标记。取决于其对比度的平方其散射长度的密度和该其他部件选择的部件或区域的之间的复合软物质或生物物理形态内差异可以不化学改变系统进行的散射实验可见或不可见。此外，中子高度穿透性并且可以用作非破坏性的探针和用于特殊环境学习样本，其中来自放置在光束的另外的材料的贡献可以是RELI巧妙地测量和校正。 弹性散射实验提供关于样本的结构和形态学的信息。散射强度是在倒易空间测量动量传递Q，其中Q =4π/λ罪的函数 Θ/ 2，用λ - 中子波长和Θ - 散射角;这随后通过逆傅立叶变换转换成实空间。因此，大的Q值与短长度尺度，具有由经典中子衍射（ND）调查了原子间的相关性。在小Q值，大尺度可以通过小角度中子散射（SANS）进行探讨。通常情况下，单个或组装在溶液合成或天然高分子，熔融，膜或大块样品的特征在于在一个宽长度尺度，从纳米和微米尺寸，通过经典的针孔SANS的应用和ULTRA-SANS（基于聚焦或单晶衍射法）的技术。然而，不同的方法或设施用于实现一个完整的结构表征的组合是由于诸如样品的可用量，在长的时间尺度样品稳定，在特殊的热力学条件的影响的再现性，和实验数据的联合分析的问题，有时难以在不同的实验几何获得。此外，随着结构和特点是高空间或时间分辨率快速结构性变化应对的研究是非常具有挑战性的，需要非常特殊的实验装置。因此，高度灵活的SANS仪器，其中限制可以超越典型的配置在一个简单而可行的方式推动发展，是满足来自用户社区的所有特殊要求是有益的。 SANS的衍射KWS-2（ 图1）中 ，歼操作2，巫妖中子科学（JCNS）在亨氏迈尔-莱布尼茨中心（MLZ）在嘉兴的中心，本来是一个经典的针孔SANS仪器从高中子通量受益的FRM II中子源交付（ 补充图1）1专用导向系统2-4。反复升级之后，该仪器是为一个宽Q系列，勘探优化，1×10 -4和0.5之间埃-1，提供高的中子强度和可调节的分辨率。在具体的样品环境和辅助设备（ 见表1）的可用性，该仪器可以配到研究软物质，并在一个很宽尺度生物系统，从纳米到微米，通过静态测量;它也可以在平衡或下变换由于动力学过程进行结构和形态的时间分辨的调查，跨越分钟几十之间的范围的时间尺度毫秒。在常规工作模式（ 图2A），7×10 -4 A -1和0.5埃之间使用Q范围-1可通过样品到检测器的距离及/或波长的变化范围之内。因此，从10长尺度结构层次和关联效应高达9000可以在现实空间（其中维度被视为2π/ Q）进行检查。波长，提供波长传播Δλ/λ= 20％的选择，4.5埃和20埃，使用机械单色（速度选择器）之间的准直条件的变化（准直长度L C和光圈的开口，A C -入口孔，在梁的最后一个中子引导段，和A S以下-样品孔，只在该样品的前面）和检测距离L 的D是自动完成的，通过计算机控制。 Q m和在在MHz范围内的高计数率的快速检测最近进行的，目的在于提高该仪器的性能。在此过程中，该仪器配备了附加功能。 有一个双盘遮光器5与可变狭缝开口（ 补充图2）和时间飞行（TOF）数据采集模式。斩波可以在10赫兹和100赫兹之间的变化频率f 斩波器和在0℃角之间两个斩波器窗口的角的开口进行操作≤Δφ≤通过相对于彼此改变两个盘的定位90°。在波长分辨率Δλ/λ的改进是通过缩短中子导向τ的开口时间瓦特由DECR实现宽松Δφ和/或增加˚F 菜刀 。记录在检测器上所产生的脉冲被分割为w的宽度匹配τ该和由旨在Δλ/λ的特征在于时间信道的适当数量。 也有一个直径为50毫米（ 图1）由镁萤石氟化镁抛物线透镜6的聚焦元件。 26 氟化镁透镜被分成三个包（4 + 6 + 16的透镜），其可以独立地在梁被移动以实现具有不同波长λ= 7-20的聚焦条件。为了减少对在镜片材料中声子散射以增加传输，透镜是使用特殊的冷却系统保持在70ķ。 有一个与1毫米的位置分辨率和0.45毫米像素尺寸的二次高分辨率位置灵敏的闪烁探测器。该探测器通常被放置在塔上的真空罐的顶部以固定距离Lð= 17微米，可以或缩小的光束（ 图1）垂直移动。主检测器以20米停在罐的端部位置，而当正在执行使用透镜高分辨率调查（低Q）4,7-次级检测器中束移动。次级检测器被放置在透镜系统的一个焦点，而在L C的小入口孔径= 20 M.将，在这种情况下，在另一个焦点。 有一个由144 3他管的阵列的一个新的主检测系统（每个为85％λ= 5的管子的全球效率）和限定有源检测区域相当于0.9米2（ 图1）。安装在一个封闭的情况下，在3他管架的背侧创新快速读取电子提高了读出的特点和降低背景噪声。取代了旧闪烁检测器（6李闪烁体和8x8光电倍增管的阵列， 图1）的新系统的特点是25毫微秒的有效死区时间常数和一个总体计数率高达5兆赫在10％死区时间用于平面曲线。这些特征是由于该系统包含在并行操作的独立的信道的事实，这是在该事件后遇到的死区时间的系统的优点。高得多的计数率缩短了测量时间，因此增加了可以在同一时间段进行的实验次数。 与所有这些创新，仪器成为一种高度通用的工具，可以通过提供可以被选择，并在一个直接和用户友好的方式使用多种工作模式（ 表2）满足广泛的结构研究。在高强度模式（图2B），最多十二倍的强度的增益相比，在相同的分辨率的现有针孔模式可以用透镜通过增加样本大小来实现。在斩波和TOF数据采集可调分辨率模式，改进的不同Q范围内的散射特征的表征是由可能性启用以改变2％和20％5之间的波长分辨率Δλ/λ。在更多的问答 -range模式（ 图2C），使用透镜和次级高分辨率检测器，且Q m低至1×10 -4 -1可以实现的，其中，在结合针孔模式，许可证尺寸从纳米到微米范围内的勘探在连续长度尺度。利用对于缩小Δλ一菜刀/λ通过避免重力和色彩效果WH提供准确的光束特性连接使用的镜头。在实时模式中，通过利用高强度和样品中进行数据采集的外部触发，结构的变化可以用下降到50毫秒时间分辨率解决。通过用斩波提高波长分辨率下降到Δλ/λ= 5％，如2毫秒一样好时间分辨率可以实现。 这里，我们提出在详细描述如何典型实验在其不同的工作模式，以及如何能够从通过数据减少所收集的数据来获得从所研究的样品的结构信息的KWS-2进行的协议。在本演示中，我们将使用SANS表征，以标准粒子溶液多种尺寸和一个高度浓缩的聚合物胶束解决方案，以显示如何大小，顺序可以在被研究过广泛的范围与KWS-2灵活高效的方式1实验性会议。聚苯乙烯球形颗粒迪菲租金尺寸（R = 150，350，500，1000，和4000的半径）和σR的尺寸分散度<img alt="公式5" src="/files/ftp_upload/54639/54639eq5.jpg" /> 8％的分散在水溶液以1％的体积分数（90％D 2 O和10％H 2 O的混合物）。以12％的浓度在D 2 O中由C 28 H 57 -PEO5嵌段共聚物形成的胶束显示出有序结构。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>heavy water D2O</td>
			<td>Sigma-Aldrich</td>
			<td>151882</td>
			<td></td>
		</tr>
		<tr>
			<td>heavy water D2O/H2O</td>
			<td>Sigma-Aldrich</td>
			<td>151882</td>
			<td>90% D2O and 10% H2O</td>
		</tr>
		<tr>
			<td>3000 Series Nanosphere Size Standards (polystyrene)</td>
			<td>Thermo Scientific</td>
			<td>3030A</td>
			<td>90% D2O and 10% H2O</td>
		</tr>
		<tr>
			<td>3000 Series Nanospher Size Standards (polystyrene)</td>
			<td>Thermo Scientific</td>
			<td>3070A</td>
			<td>90% D2O and 10% H2O</td>
		</tr>
		<tr>
			<td>3000 Series Nanosphere Size Standards (polystyrene)</td>
			<td>Thermo Scientific</td>
			<td>3100A</td>
			<td>90% D2O and 10% H2O</td>
		</tr>
		<tr>
			<td>3000 Series Nanospher Size Standards (polystyrene)</td>
			<td>Thermo Scientific</td>
			<td>3200A</td>
			<td>90% D2O and 10% H2O</td>
		</tr>
		<tr>
			<td>3000 Series Nanosphere Size Standards (polystyrene)</td>
			<td>Thermo Scientific</td>
			<td>3800A</td>
			<td>90% D2O and 10% H2O</td>
		</tr>
		<tr>
			<td>diblock copolymer C28H57-PEO5k</td>
			<td>synthesized in house</td>
			<td></td>
			<td>in D2O</td>
		</tr>
		<tr>
			<td>Quartz Cells 110-QX</td>
			<td>Hellma analytics</td>
			<td>110-1-46</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum cuvette-holder</td>
			<td>manufactured in house</td>
			<td></td>
			<td>for measurements at ambient temperature</td>
		</tr>
		<tr>
			<td>screwdriver</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allen keys</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

studying soft-matter and biological systems over a wide length-scale from nanometer and micrometer sizes at the small-angle neutron diffractometer kws-2

该KWS-2 SANS衍射仪是专门为软物质和覆盖面广长度尺度，从纳米到微米的生物系统调查。该仪器通过组合古典针孔，聚焦（用透镜）为广动量传输Q范围的勘探优化1×10 -4和0.5埃-1之间，并且（与斩波器）的方法飞行时间- ，同时提供高-neutron强度具有可调分辨率。由于调整强度和在实验过程中在宽范围内的分辨率来装备特定样品的环境和辅助设备的能力，具有的可能性相结合，所述KWS-2示出了在处理的广泛范围的结构和形态学研究的较高的通用性场。平衡结构可以在静态测量进行研究，而动态和动力学过程可以在调查分钟之间的时间尺度到几十毫秒哔声以时间分辨的方法。与该KWS-2盖调查从表现出多种结构层次（如凝胶，网络或团聚体），以小而差的散射系统（如单聚合物或蛋白质复杂，分级制度的范围内典型的系统解）。最近检测系统，这使得在MHz范围内计数率的检测升级，开辟新的机会来研究接近在高Q缓冲散射水平弱散射信号缓冲溶液中即使很小的生物形态。 在本文中，我们提供了一个协议，以调查与特征尺寸水平跨越了广泛的长度规模和参展使用KWS-2中尺度结构订购样品。我们提出具体如何使用由该仪器所达到的性能水平提供了多种工作模式。

Watch this Scientific Journal Video about Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2 at Jo

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

在小角度中子衍射仪KWS-2研究软物质和生物系统在广泛的长度尺度从纳米，微米尺寸

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

studying-soft-matter-biological-systems-over-wide-length-scale-from

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.2K 次观看.  Forschungszentrum Jülich GmbH. Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to µm that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

视频: Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

科研

生物工程

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

小型和广角X-射线散射研究生物大分子的解决方案

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different techniques and varies in price, materials, and purpose. Many of these fabrication methods result in polydisperse populations with non-uniform activation thresholds. Additionally, controlling the droplet sizes typically requires stable perfluorocarbon liquids with high activation thresholds that are not practical in vivo. Producing uniform droplet sizes using low-boiling point gases would be beneficial for in vivo imaging and therapy experiments. This article describes a simple and economical method for the formation of size-filtered lipid-stabilized phase-shift nanodroplets with low-boiling point decafluorobutane (DFB). A common method of generating lipid microbubbles is described, in addition to a novel method of condensing them with high-pressure extrusion in a single step. This method is designed to save time, maximize efficiency, and generate larger volumes of microbubble and nanodroplet solutions for a wide variety of applications using common laboratory equipment found in many biological laboratories.

This protocol describes a method of generating large volumes of lipid encapsulated decafluorobutane microbubbles using probe-tip sonication and subsequently condensing them into phase-shift nanodroplets using high-pressure extrusion and mechanical filtration.

从预成型微气泡生产膜过滤相移十氟丁烷纳米滴

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

使用无细胞系统快速表征遗传部分

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...