Name: microfluidic chips for in situ crystal x-ray diffraction and in situ dynamic light scattering for serial crystallography
Uploaded: 2018-04-24T13:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography at JoVE.com

这项工作得到了码头种子基金 PIF-2015-46、BMBF 赠款05K16GUA 和05K12GU3 的支持, 以及 "汉堡超快成像中心--德意志原子能规模卓越集群中的物质结构、动力学和控制"。Forschungsgemeinschaft (DFG)。与自由电子激光科学中心相关的作者的工作由亥姆霍兹协会通过方案导向基金提供资金。同步辐射 MX 数据是收集在光束线 P14 经营的 EMBL 汉堡在佩特拉 III 储存环 (DESY, 汉堡, 德国)。

1. 芯片设计和主制造
<ol><li>面具设计<ol>
<li>使用合适的 CAD 绘图程序勾勒出所需的通道几何图形。对于每个光刻胶层, 准备一个单独的掩码。本协议中使用的所有设计都将在结果部分详细讨论, 并可作为 AutoCad 提供。DWG 在辅助文件 1中的格式。 注: 对于构建所有的多路设备, 通道的高宽长宽比不应超过 1:10, 以防止通道崩溃。聚酰亚胺箔和固化环氧树脂是更加坚固和原则上应该也允许更高的纵横比。然而, 我们故意不超过1:10 的比例, 使最初的设计可以原型作为传统的。</li>
		<li>将 CAD 文件翻译成乳化膜光掩膜。使用标称的 64k DPI 分辨率, 使精确的功能降低到大约5µm 大小。 注意: 这可以通过商业服务来完成。成像服务可能更喜欢不同的绘图约定, 以简化掩码图像的文件转换。在转换过程中, 请先查询首选的绘图约定, 以避免费力的故障排除。在黑色背景上具有透明功能的面具将图案 SU8 光刻胶的功能到晶片上, 以产生功能性的微在复制品成型。反过来, 透明背景面具上的黑色特征需要准备适合 X 射线芯片制作的注塑模。我们建议同时订购两个掩码极性, 以便在将设计转换成 x 射线芯片之前, 先制作和设计验证, 以便制造出更多的产品。</li>
	</ol>
</li>
	<li>SU8 大师制作 注意: 这是唯一需要在洁净室中进行的过程。如果关键的洁净室设备不可用, 整个步骤可以外包给 MEMS 铸造服务公司, 提供现成的图案 SU8 大师。根据数据表指令处理 SU8。1.2.1 到1.2.4 的步骤总结了一般 SU8 主制造工作流, 并列出了表 1中所列的三层 X 射线芯片设计的完整参数。已在以前的29中发布了多层 SU8 对齐的介绍。<ol>
<li>使用适当的旋转速度和时间, 在表 1 (图 1, 步骤 1) 中, 倒约1毫升的 SU8 抵抗到3英寸的晶片和旋转外套 SU8 到所需的厚度。根据层厚度在65摄氏度和95摄氏度前烘烤光刻胶, 每分钟几分钟。执行预烘烤, 以巩固 SU8, 以防止它坚持光掩模和提高抗粘附于基体(图 1, 步骤 2)。</li>
		<li>将光刻胶暴露在表 1中指定的 UV 光中, 后跟曝光后烘烤到95摄氏度, 催化完成曝光期间启动的 photoreaction (图 1, 步骤 3)。</li>
		<li>对每个后续层重复这些步骤。然后使用掩码光刻和游标卡尺对齐标记29将后续图层的光掩膜与主控形状对齐。</li>
		<li>冲洗所有未曝光的 SU8 抵制通过开发的晶片在丙烯乙二醇甲醚醋酸 (PGMEA), 直到异丙醇冲洗不再显示乳白色沉淀 (图 1, 步骤 4)。用加压氮气干燥晶片。 注: 异丙醇是 SU8 的一种不良溶剂, 其降水表明残留的成灾仍然存在。</li>
	</ol>
</li>
	<li>模具制造<ol>
<li>将一块铝箔 (15 x 15 厘米) 放入培养皿中 (10 厘米), 并将 SU8 主放到铝箔上, 使其易于去除。</li>
		<li>将硅胶底座与固化剂 (10:1) 混合, 产生总用量25克, 用铲罐或机械搅拌机大力搅拌。一个3英寸的晶片在培养皿 (10 厘米) 消耗约25克的, 以导致5毫米厚板。</li>
		<li>将预混合的 SU8-master (图 1, 步骤 5) 倒到4毫米的高度. 脱水为5分钟, 以去除气泡直到没有, 或者只有很少的气泡留在了表面。</li>
		<li>在烤箱中以70摄氏度的温度治疗1小时。然后用手术刀切开固化的 SU8, 然后从主控形状 (图 1, 步骤 6) 中轻轻剥离该模型。切断所有的方式到主, 以防止在剥离过程中的硅烷裂纹。</li>
		<li>可选: 准备截面片的注塑模具, 以确认所有 SU8-layers 在主具有所需的厚度 (图 1)。在所有的芯片中都可以进行早期微流控通道布局测试。 注: 使用二十年代、0.4 毫巴 o2、50 W、13.56 兆赫, 通过 O2等离子激活, 在冲压接入端口 (步骤 3.3) 后, 可将复制模塑料直接粘合到玻璃基板上。如第1.2 节所述, 这需要相反的掩模极性, 因此, x 射线芯片制造的晶片轮廓。</li>
	</ol>
</li>
</ol>2.原位X 射线芯片制作
<ol><li>在乙醇中稀释两种环氧树脂前体, 最终乙醇浓度为 40 wt%。在乙醇中, 每一个环氧树脂前驱体的总质量为0.25 克, 足以用于1厘米的2芯片。 注: 这降低了粘度的产生5分钟环氧树脂, 以简化无气泡混合和复制模铸造, 并尽量减少最终固化环氧树脂层的厚度。乙醇在固化步骤中蒸发通过了该硅烷。</li>
	<li>在真空干燥中加以30分钟, 使其在成型过程中能吸收环氧树脂中的小气泡。</li>
	<li>将聚酰亚胺箔切割到大约 70 x 70 毫米, 并用胶带在 75 x 50 毫米的玻璃上滑动, 以获得平面和刚性表面与背面的胶带。等离子激活箔与 50 W, 13.56 兆赫, 0.4 毫巴 O2血浆为二十年代, 然后孵化完全箔滑在水溶液中1体积% (3-氨基丙基) 三氯 (APTS) 或 (3-glycidyloxypropyl) 三氯 (GPTS) 5 摄氏度在20°c。</li>
	<li>彻底混合乙醇稀释环氧树脂前体溶液, 确保最佳固化行为。从真空室中提取注塑模, 并将其放置在平坦的表面上。然后, 用一个微 (大约10µL 1 厘米的显微组织) ( 图 1, 步骤 7a), 快速地将一滴混合树脂放到模具上的每个显微结构上。</li>
	<li>从水硅烷 (APTS 或 GTPS) 溶液中提取聚酰亚胺箔滑动三明治。用加压的空气或氮气烘干箔。</li>
	<li>将准备好的聚酰亚胺箔-玻璃滑动三明治放到沉积环氧树脂上 (图 1, 步骤 7b)。将玻璃滑块紧压在聚酰亚胺箔上, 对抗该模。将金属片放在玻璃滑块上, 然后将重量放置在1.4 厘米/cm2压力为1小时, 而环氧树脂固化室温。 注: 理想情况下, 在模具中的结构具有最大高度的区域, 箔上没有树脂残留。这些对应于结晶井之后结晶发生。<ol>
<li>可选: 如果小特性的精确成型是关键的, 在成型步骤31中, 可以用铝框架增强该塑料模具。</li>
	</ol>
</li>
	<li>用聚酰亚胺箔和带图案的环氧树脂去除玻璃滑块, 将其从注塑模中剥离 (图 1步骤 8)。等离子体激活带图案的环氧侧与 50 W, 13.56 兆赫, 0.4 毫巴 O2等离子二十年代。</li>
	<li>将聚酰亚胺箔从等离子腔中除去后, 在1体积% 水 APTS (或 GPTS) 溶液中孵化出环氧树脂图案的箔, 5 分钟20摄氏度。同样, 用互补的1体积% GPTS (或 APTS) 硅烷活化制备第二个未图案的聚酰亚胺箔。孵化后, 干燥的结构和非结构化箔与加压空气。</li>
	<li>将环氧侧面朝上放在平坦的表面上, 使用下面的水滴作为介质的表面张力, 防止箔卷曲并确保最大平整度。然后将第二活化聚酰亚胺箔放在顶部, 轻轻地用手指从一个角向相反的方向条纹, 使它们结合起来, 避免气泡的形成。</li>
</ol>3. 流体输送用端口
<ol><li>根据1.3.1 到1.3.3 的步骤, 在不使用 SU8-master 的情况下, 在培养皿中准备4毫米厚的板。将板材切成合适尺寸的基板, 以覆盖芯片中的所有入口端口, 而不覆盖芯片的单个结晶隔间。</li>
	<li>等离子激活两个, 芯片和在 50 W, 13.56 兆赫, 0.4 毫巴 O2等离子二十年代。对于化学键合, 然后在1体积% 水 APTS 或 GPTS 溶液中孵化每个部分, 5 分钟20摄氏度。用加压的空气干燥每个部件, 然后将其板压在箔片上。</li>
	<li>为了改善粘合, 将芯片放在平板上, 然后用塑料箔盖住, 接着是一个干净的玻璃滑梯和一块金属块。最后, 将1.4 的 N/厘米2压力应用到大约1小时的存款权重。</li>
	<li>在芯片设计中标记了入口和出口端口的每个位置都有一个0.75 毫米的穿孔孔, 并用胶带封住背面。该芯片现在可用于任何直径与孔直径匹配的油管 (在步骤4.2 中详细说明)。</li>
</ol>4. 表面处理
<ol><li>在氟溶剂中准备1:20 稀释 9 wt% 氟聚合物, 最终浓缩为 0.45 wt%。储存的股票解决方案和稀释在一个冰箱在黑暗中的4摄氏度。</li>
	<li>将1:20 氟聚合物稀释成1毫升鲁尔锁注射器。将 27G x 5/8 "针连接到注射器上, 然后将聚四氟乙烯导管贴到针上。</li>
	<li>将油管连接到 X 射线芯片插座, 然后在步骤4.1 中注入含氟聚合物工作溶液, 直到所有通道都填满为止。</li>
	<li>将晶片与平边放在190摄氏度热板上, 5 分钟蒸发所有溶剂, 将氟聚合物存入薄膜涂层。 注: 使用新几何时, 检查通道是否在涂层过程中被含氟聚合物堵塞。如果是, 进一步稀释库存解决方案。</li>
</ol>5. 蛋白质制备
<ol><li>权衡冻干奇异果甜蛋白并将其溶解在表2中列出的缓冲区解决方案中, 以获得40毫克mL-1 的最终蛋白质浓度。</li>
	<li>根据制造商协议, 在表 2中列出的缓冲区上透析葡萄糖异构酶。</li>
	<li>按照舒伯特et.的描述, 准备蛋白质 thioredoxin。30。</li>
	<li>使用表 2中总结的消光系数 (由软件 ProtParam32计算) 验证最终蛋白质浓度测光地。</li>
	<li>使用超纯水制备所有溶液, 并用0.2 µm 过滤器过滤。</li>
	<li>离心蛋白溶液在20°c 15 分钟 16100 x g, 并采取上清结晶实验。</li>
</ol>6. x 射线芯片中的蛋白质结晶
<ol><li>在微流控芯片中结晶蛋白质, 混合等量的蛋白质溶液和沉淀溶液。在表 2中总结了蛋白质浓度、缓冲成分和沉淀组成。准备一个总体积约20µL, 以填补微流控芯片。</li>
	<li>混合后, 立即通过注射器将溶液注入芯片的入口端, 再加上 27G x 5/8 "针和聚四氟乙烯油管, 外径为0.75 毫米 (详见步骤 4.2)。 注: 串行布局的灌装程序需要事先启动的芯片与含氟油, 这是最简单的, 从出口港口加载氟化油之前, 通过进口端口注入结晶溶液。所有加载步骤都应使用显微镜监测, 以控制应用的注射器压力和相应的流速。</li>
	<li>在填充芯片后, 通过将含氟油注入芯片的入口端, 将单独的结晶隔间分开。通过阻止芯片的所有入口和出口端口来密封芯片。这可以通过插入回形针来完成。 注: 由于结晶室由蛋白质/沉淀溶液填充, 氟化油只填充芯片的入口通道, 不影响结晶室中的溶液。</li>
	<li>为了模拟气相扩散结晶动力学, 将密封芯片置于常温和正常大气中, 使结晶室中的水滴通过聚酰亚胺箔的蒸发而收缩。</li>
	<li>通过显微镜或 dl 测量 (步骤 7) 观察晶体形成后, 将完整的微流控芯片转移到适当的沉淀溶液中, 停止结晶井的进一步蒸发, 直到 x 射线衍射实验进行。</li>
</ol>7. 晶片结晶井的动态光散射测量
注意: 用100兆瓦的激光输出功率进行 dl 测量, 波长为 660 nm, 在142°的散射角检测到散射光。因为所有被调查的样品解答是水的折射率 (n = 1.33) 在所有演算使用了。
<ol><li>使用步骤8.1 中描述的适配器将微流控芯片置于 dl 仪器的 SBS 格式板持有者中。将适配器插入设备。</li>
	<li>使用机动的 x、y、z 级, 仔细调整微流控芯片内的激光焦点。由于微流控芯片非常薄, 所以应用小增量步骤调整 z 电平。 注意: 一个正确的调整是由高截距和平滑尾部确定的, 由此产生的自相关函数的 dl 测量。可以创建一个校准文件, 以匹配每个单独的结晶井在微流控芯片中的位置, 允许随着时间的推移, 在几个舱室自动 dl 测量。</li>
	<li>执行每一个 dl 测量在 293 K 三十年代和重复测量每5分钟, 直到结束的结晶实验。 注: 初始成核可以跟随 dl 测量的半径分布, 随着时间的推移, 成功的晶体形成可以并行, 其次是在内置显微镜的 dl 板阅读器。</li>
</ol>8. 衍射数据收集
<ol><li>用于光束线测角仪的适配器<ol>
<li>打印量角器板的适配器, 在晶体数据收集过程中定位和旋转 X 射线芯片。</li>
		<li>使用制造商推荐的默认参数设置, 在爱好级别3D 打印机上为基量角器制造适配器。 注意: 适配器是使用3维 CAD 系统设计的, 适配器的坐标文件被附加在 "" 中。STL 文件格式的补充。</li>
		<li>使用双面胶带将 X 射线芯片固定到适配器上。</li>
	</ol>
</li>
	<li>
原位x 射线晶体学<ol>
<li>使用 10 x 5 µm (高斯剖面 FWHM) 的光束大小收集衍射数据 296 k 使用 X 射线以能量12.8 凯文和通量2.2 ·1011光子·s-1在衰减光束和记录衍射模式使用 Pilatus 6M 混合像素探测器。 注: 含有奇异果甜蛋白、葡萄糖异构酶或 thioredoxin 晶体的微流控装置用于原位 X 射线晶体实验, EMBL 光束线 P14 的佩特拉 III. 同步加速器。在其他 X 射线源中, 可用光束聚焦大小和通量可能不同。在表 3中总结了暴露的蛋白质晶体数量、每个晶体中记录的衍射图案数、每个曝光的振荡角度范围和曝光时间。</li>
		<li>分别使用程序 XDS33的两个连续衍射模式的进程集。使用 xds 中发现的 bash 脚本 "sh"。</li>
		<li>从所有晶体中创建每个数据集的 HKL 文件, 并使用软件嵌入式33来缩放它们。在补编中使用 bash 脚本 "嵌入式" 来创建嵌入式的输入文件。 注意: 只有具有大于90% 的相关系数的晶体数据集才能进行缩放。保守的标准‹I/σ (I) › (> 2) 应该被用来确定最高分辨率壳。使用程序 MOLREP34从 CCP4 套件35中进行的分子替换可用于通过使用表 3中显示的蛋白质数据库 (PDB) 的3D 坐标来获取进一步模型生成的阶段。</li>
		<li>使用 Refmac535、36 isotopically 优化所有结构, 并使用傻瓜37对最终模型进行可视化检查。 注: 在细化过程中应自动添加溶剂分子, 需要检查以确定化学合理的位置。必须检查所有模型以识别拉玛钱德朗异常点。</li>
	</ol>
</li>
</ol>9. 数据评估
<ol><li>辐射损伤<ol>
<li>使用欧文et al38描述的方法分析衍射功率随时间衰减的情况。为此, 计算每个计算的衍射数据集 (2 连续衍射模式) 的所有索引反射的 XDS (i) (由33提供) 的总总和, 以用作参考值。使用 "ISigma" 的 bash 脚本从补充。</li>
		<li>将每个数据集的衍射功率正常化为第一个数据集的平均衍射功率。</li>
		<li>通过从 XDS33 (bash 脚本 "Rmeas. sh") 中获得的正确. LP文件中的 Rmeas 值, 分析 Rmeas 值随时间变化的情况。</li>
	</ol>
</li>
	<li>水晶方向<ol>
<li>确定欧拉角, 获取关于实验室坐标系的晶格方向分布的信息。使用软件 Matlab 计算输出文件 XPARM39中给定的 XDS 方向矩阵的欧拉角。使用 bash 脚本 "rotation_matrix" 从 XPARM 文件中提取每个晶体的旋转矩阵。在 matlab 中使用输出文件作为输入, 用 matlab 函数 rotro2eu (补充文件) 计算欧拉角。 注意: 计算的详细说明已由 Zarrine-Afsar et al40发布。</li>
		<li>将获得的欧拉角从弧度转换为度数。将所有三旋转平面 (xy、xz 和 10°) 在类中获得的欧拉角分组, 并用软件 Origin9 进行绘制。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有什么可透露的。

以环氧树脂为填充材料, 聚酰亚胺箔为窗口材料, 制备了用于原位X 射线衍射的微流控器件。我们的程序通过以前的 X 射线芯片设计16,21, 优化了制造过程的各个步骤。我们减少了窗口的厚度, 从而使背景散射, 同时也放宽制造, 因为需要更少的工艺步骤。使用描述的协议进行原位结晶具有很大的好处。它允许在室温下进行衍射数据收集, 从而排除了低温保护的需要, 在某些情况下, 它包含了在蛋白质结构中引入人工制品的风险。此外, 晶体不受物理应力的作用, 因为可以避免晶体从其原生态环境中转移。通过这个过程, 晶体保持其最高的质量, 不遭受任何治疗。
根据我们的经验, 协议中最关键的步骤围绕着控制结晶过程。获得合适尺寸的 X 射线合适晶体的参数需要进行经验主义的鉴定, 不能直接从蒸气扩散实验中提取。使用相同浓度的蛋白质和沉淀并不总是导致晶体在不同的芯片, 或有时在不同的油井内相同的芯片。这表明, 所有影响晶体成核和生长的因素都应仔细考虑, 如母液成分或结晶动力学 (通过蒸发轨迹)。当更大的水晶衍射对更高的决议, 适当地大水晶是理想地增长。晶体成核和生长过程可以跟随 dl 测量。调整激光对焦内的50µm 薄结晶室的芯片可能是一个挑战, 可能需要仔细手动对准。采用深度大于100µm 的井眼, 激光自动对准是可行的、可靠的, 可以通过自动采集方案对多井进行监测。
基于聚酰亚胺的 x 射线芯片只产生低背景, 我们通过求解三模型蛋白结构, 证明了这些器件对常规 X 射线衍射数据的适用性。与以前实现的分辨率相比, 在芯片中获得的最佳分辨力与以往的分辨率不同, 从显着较大的蛋白质晶体和常规的 X 射线数据收集。这可能是由于几个因素, 进一步的结晶条件优化可能进一步提高衍射。可以将原位衍射数据收集到1.8 Å分辨率, 应用晶体尺寸小于30µm。对奇异果甜蛋白衍射数据的详细分析提供了对辐射损伤的见解。为了限制辐射损伤的扩展, 微流控装置中每个隔间只有一个单晶, 因为自由基的扩散会发生在相邻的晶体中。为了提高数据收集的速度, 将来应该自动化。
由于晶体形态, 在某些情况下, 可能会出现首选方向。这是例如案件与 thioredoxin 数据集, 水晶有一个强烈优选的取向相对于芯片窗口。即使在这里, 我们也可以收集一个完整的衍射数据集。如果晶体在芯片中表现出一个首选的方向, 特别是如果相应的空间群也有一个低对称性, 那么数据集的完整性应该在收集过程中被监视, 这样足够的衍射模式收集。
使用这些芯片的时间分辨研究是直接可能的, 当利用光诱导反应与泵探针方法。为泵浦激光器的聚酰亚胺箔光传输需要阐明, 或者, 可以使用光学上清楚的聚酰亚胺或环烯烃。目前的微流控几何不允许在晶体生长后的基质混合实验。然而, 我们期望所描述的 x 射线芯片制造协议也适用于这种混合设计的时间分辨 X 射线衍射以及散射方法19。

了解蛋白质的3D 结构对于理解其功能至关重要。近原子分辨率结构是迄今为止最常见的 X 射线晶体学。该技术将蛋白质晶体暴露于 x 射线辐射中, 进而分析了产生的衍射模式, 用于结构的确定和细化。在传统的 x 射线晶体学中, 一个完整的衍射数据集是从一个单一的, 理想的大, 在低温温度的晶体记录。然而, 这种晶体大多不是微不足道的生长, 并确定适当的冷冻保存条件本身可能会变得具有挑战性, 有时也可能导致偏差从原生蛋白结构5。
最近在 x 射线自由电子激光器和同步辐射同步辐射的技术进步已经允许解决结构从较小的晶体, 作为新的微聚焦同步辐射, 增加 x 射线光束的辉煌, 和改进的 x 射线探测器成为可用的6,7。通常, 小晶体比大的和缺陷的自由晶体更容易生长8,9。然而, 小晶体遭受 X 射线辐射损伤比大晶体快得多。这是因为比起一个大的晶体, 更高的 X 射线剂量必须投射到一个较小的晶体体积, 以衍射可比分辨率。因此, 即使是低温保护也往往不足以记录一个完整的衍射数据集从一个单一的晶。
为了克服这一障碍, 串行晶体学已成为从许多随机定向的纳米微晶中收集和合并衍射图案以获得完整数据集的选择方法。辐射引起的晶体损伤最小化, 通过传播用于解决高数量晶体的蛋白质结构的总 X 射线剂量5,10。在一个 ' 衍射之前销毁 ' 自由电子实验, 每个晶体只用于一个曝光使用飞第二 X 射线脉冲。第三代同步辐射源的微聚焦同步辐射反过来可以执行串行晶体学, 其时间为几毫秒短 X 射线曝光11,12,13,14。但是, 在数据收集过程中, 如果没有晶体振荡或旋转, 则只能记录部分布拉格反射, 因此结构确定15通常需要数以万计或更多的衍射模式。到目前为止, 已为串行晶体学开发了一系列不同的样例传递方法, 最近已回顾了14、16、17、18、19。其中, 在 X 射线曝光期间, 几个固定目标的样本交付策略成功地与晶体旋转结合在一起, 从而大大减少了衍射模式, 可以提供同样完整的数据集, 同时消耗较少样品与经典的系列结晶学实验相比, 仍然有图像被记录7,16,20,21,22,23,24。
提出了一种利用低 x 射线背景制备微流控器件的协议。该装置采用软光刻法从5分钟的环氧树脂胶中进行花纹, 适用于室温下的原位x 射线衍射实验, 其结果是将样品制备直接纳入 x 射线的设置中, 如经过时间分辨的研究, 遵循混合诱导的动力学18,19。微流控通道是盖在两侧与聚合物聚酰亚胺箔, 导致 x 射线窗口与联合厚度约16µm, 允许低 x 射线背景成像。所有使用的材料都提供良好的耐溶剂性。这种制作方法比较简单, 价格低廉。在 SU-8 主晶片的采购后, 所有的制造都可以在一个洁净室内完成一个典型的研究实验室设置。
在一个应用示例中, 我们描述了基于量角器的固定目标序列晶体学的芯片。首先, 讨论了用毛细管阀门 microfluidically 将水反应分解成选定数量的 nanoliter 液滴的设计和制作注意事项。这种加载机制避免了通道死容量的样本损耗, 并且可以在不使用泵或其它液体驱动设备的情况下手动进行拆分。通过动态光散射 (dl) 控制蛋白质晶体的成核和生长, 对这种孤立的 nanoliter 大小的蛋白溶液进行了监测原位。以前已经证明, dl 测量可以在微流控设备上执行, 该装置由连接到玻璃滑块25、26的烷结构组成。由于聚酰亚胺层具有比 550 nm 长的波长的高传输, 因此在使用适当的激光波长27、28时, 该方法可以扩展到 X 射线透明芯片中的测量。在 dl 结果的基础上, 可以观察到初始成核, 并可以停止进一步的雾滴蒸发以获得较少但更大的蛋白质晶体。
在足够的晶体生长后, 可以使用基于量角器的原位固定目标序列 x 射线晶体学在室温下收集完整的 x 射线衍射数据集。使用一套软件工具和定制脚本处理衍射数据集, 以解决蛋白质晶体结构。这种技术避免了传统晶体学实验中常用的低温保存过程中产生的人工制品。
我们比较了三个蛋白质靶结构, 解决使用约10-20 µm 小晶体生长在芯片, 更好, 然后2Å分辨率。通过结晶和衍射原位, 处理和因此脆弱的晶体的机械扰动最小化。该协议适用于衍射高分辨率和低分辨率的蛋白质晶体 (1.7 Å到3.0 Å)。几乎每一个晶体都可以用于衍射, 小样本被浪费了, 这使得这是一个非常有效的水晶传递方法。
本协议提供了如何为原位蛋白质结晶和衍射数据收集准备 X 射线透明微流控芯片的详细指南。该程序是精心设计的, 以得益于微流控精度, 而不需要先进的设备在实验室。此外, 在同步加速器光束线的数据收集可以不需要专门的量角器或加湿器来缓解非专家的结果再现。所提出的技术可应用于室温下的串行毫秒结晶学数据采集, 同时保持辐射损伤最小, 不引入低温保护或晶体处理对晶体生长后的应力。因此, 所述方法适用于任何蛋白质结晶项目。

<table><tbody><tr><td>SU-8 3000 Series</td><td>MicroChem Corp.</td><td>SU-8 3000</td><td>Photoresist</td></tr><tr><td>PGMEA</td><td>Sigma-Aldrich</td><td>484431</td><td>Developer</td></tr><tr><td>Isopropyl alcohol</td><td></td><td></td><td>Solvent</td></tr><tr><td>Ethanol</td><td></td><td></td><td>Solvent</td></tr><tr><td>Epoxy glue</td><td>UHU</td><td>Plus Schnellfest 5 min</td><td>Epoxy glue</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184</td><td>Silicone</td></tr><tr><td>Kapton foil</td><td>Dupont/ American Durafilm</td><td>HN grade, gauge 30 (7.5 &amp;mu;m)</td><td>polyimide foil</td></tr><tr><td>APTS</td><td>Sigma-Aldrich</td><td>440140</td><td>Chemical</td></tr><tr><td>GPTS</td><td>Sigma-Aldrich</td><td>440167</td><td>Chemical</td></tr><tr><td>Cytop CTX-109AE</td><td>Asahi Glass Co. Ltd</td><td>Cytop CTX-109AE</td><td>Cytop fluoropolymer coating</td></tr><tr><td>CT-Solv 100E</td><td>Asahi Glass Co. Ltd</td><td>CT-Solv 100E</td><td>Cytop fluoro-solvent</td></tr><tr><td>HFE-7500</td><td>3M</td><td>Novec 7500</td><td>Fluorinated oil</td></tr><tr><td>AutoCAD</td><td>AutoDesk Inc.</td><td>AutoCAD</td><td>CAD Software</td></tr><tr><td>Biopsy Punch</td><td>Harris</td><td>Uni-core 0.75 mm</td><td></td></tr><tr><td>Photo mask</td><td>JD Photo Data</td><td></td><td></td></tr><tr><td>3 inch wafer</td><td>University Wafer</td><td></td><td>Silicon wafer</td></tr><tr><td>Mask aligner</td><td>S&amp;Uuml;SS MicroTec</td><td>MJB4</td><td>Mask aligner</td></tr><tr><td>PDMS mixer</td><td>Thinky</td><td>ARE-250</td><td></td></tr><tr><td>Plasma machine</td><td>Diener electronic</td><td>Zepto</td><td></td></tr><tr><td>Thaumatin</td><td>Sigma Aldrich</td><td>T7638</td><td>Protein</td></tr><tr><td>Glucose Isomerase</td><td>Hamton Research</td><td>HR7-102</td><td>Protein</td></tr><tr><td>Bis-Tris</td><td>Sigma Aldrich</td><td>B9754</td><td>Chemical</td></tr><tr><td>Sodium Tartrate</td><td>Merck</td><td>106664</td><td>Chemical</td></tr><tr><td>Tris-HCl</td><td>Sigma Aldrich</td><td>10812846001</td><td>Chemical</td></tr><tr><td>HEPES</td><td>Carl Roth</td><td>6763.2</td><td>Chemical</td></tr><tr><td>Magnesium Chloride</td><td>Sigma Aldrich</td><td>208337</td><td>Chemical</td></tr><tr><td>Ammonium Sulfate</td><td>Sigma Aldrich</td><td>A4418</td><td>Chemical</td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6758</td><td>Chemical</td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>1064060250</td><td>Chemical</td></tr><tr><td>PEG1500</td><td>Molecular Dimensions</td><td>MD2-100-6</td><td>Chemical</td></tr><tr><td>SPG buffer</td><td>Jena Bioscience</td><td>CSS-389</td><td>Chemical</td></tr><tr><td>SpectroLight600</td><td>XtalConcepts</td><td></td><td>DLS Instrument</td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td></td><td>Spectrophotometer</td></tr><tr><td>Zentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Ultimaker2</td><td>Ultimaker</td><td></td><td>3D printer</td></tr><tr><td>Form2</td><td>Formlabs</td><td></td><td>3D printer</td></tr><tr><td>Amicon Filter</td><td>Sartorius Stedim</td><td></td><td>0.2 &amp;micro;m filter</td></tr><tr><td>Tubing</td><td>Adtech Polymer Engineering Ltd</td><td>Bioblock/05</td><td>PTFE tubing 0.3 mm Inner Diameter x 0.76 mm Outer Diameter</td></tr><tr><td>Syringes&amp;nbsp;</td><td>BD</td><td>309628</td><td>1ml Luer-Lock Tip</td></tr><tr><td>Needle&amp;nbsp;</td><td>Terumo Agani Needle</td><td>AN*2716R1</td><td>27Gx5/8&quot;</td></tr></tbody></table>

microfluidic chips for in situ crystal x-ray diffraction and in situ dynamic light scattering for serial crystallography

该协议描述了量角器基于固定目标序列晶体学的低 X 射线背景下制备微流控器件。该装置采用软光刻法从环氧树脂胶中进行图案化, 适用于室温下的原位X 射线衍射实验。样品井在双方盖与聚合物聚酰亚胺箔窗口允许衍射数据收集以低 x 射线背景。这种制造方法是不昂贵和廉价的。在 SU-8 主晶片的采购之后, 所有的制造都可以在实验室环境中的洁净室之外完成。芯片设计和制造协议利用毛细管阀门 microfluidically 将水反应分解成定义 nanoliter 大小的水滴。这种加载机制避免了通道死容量样本损失, 可以很容易地执行手动不使用泵或其他设备的流体驱动。我们描述如何通过动态光散射来控制蛋白质晶体成核和生长的 nanoliter 大小的蛋白质溶液的分离被监测原位。在适当的晶体生长后, 可以使用基于量角器的原位固定目标序列 x 射线晶体学在室温下收集完整的 x 射线衍射数据集。该协议提供了自定义脚本, 用于处理衍射数据集, 使用一套软件工具来解决和细化蛋白质晶体结构。这种方法避免了在常规结晶学实验中, 在低温保存或人工晶体处理过程中可能诱发的文物。我们提出和比较了三种蛋白质结构的解决使用小晶体的尺寸约10-20 µm 生长在芯片。通过结晶和衍射原位, 处理和因此脆弱的晶体的机械扰动最小化。该协议详细介绍了如何制作适合原位串行晶体学的自定义 X 射线透明微流控芯片。由于几乎每个晶体都可以用于衍射数据收集, 这些微流控芯片是一种非常有效的晶体传递方法。

Watch this Scientific Journal Video about Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography at JoVE.com

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

本协议详细描述了如何制造和操作微流控器件, 用于 X 射线衍射数据的室温采集。此外, 还介绍了如何通过动态光散射监测蛋白质结晶, 以及如何处理和分析获得的衍射数据。

用于原位晶体 x 射线衍射和原位的微流控芯片系列晶体学的动态光散射

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

microfluidic-chips-for-situ-crystal-x-ray-diffraction-situ-dynamic

Research

JoVE Journal

Chemistry

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

14.6K 次观看.  DESY. 本协议详细描述了如何制造和操作微流控器件, 用于 X 射线衍射数据的室温采集。此外, 还介绍了如何通过动态光散射监测蛋白质结晶, 以及如何处理和分析获得的衍射数据。

视频: Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

化学固定准备贴壁细胞的X射线荧光成像

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

科研

化学

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

生物化学

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

X射线粉末衍射在保护科学:迈向文物艺术品腐蚀产物的常规晶体结构测定

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

High Pressure Single Crystal Diffraction at PX^2

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the GSECARS 13-BM-C beamline at the Advanced Photon Source. The DAC program at 13-BM-C is part of the Partnership for Extreme Xtallography (PX^2) project. BX-90 type DACs with conical-type diamond anvils and backing plates are recommended for these experiments. The sample chamber should be loaded with noble gas to maintain a hydrostatic pressure environment. The sample is aligned to the rotation center of the diffraction goniometer. The MARCCD area detector is calibrated with a powder diffraction pattern from LaB6. The sample diffraction peaks are analyzed with the ATREX software program, and are then indexed with the RSV software program. RSV is used to refine the UB matrix of the single crystal, and with this information and the peak prediction function, more diffraction peaks can be located. Representative single crystal diffraction data from an omphacite (Ca0.51Na0.48)(Mg0.44Al0.44Fe2+0.14Fe3+0.02)Si2O6 sample were collected. Analysis of the data gave a monoclinic lattice with P2/n space group at 0.35 GPa, and the lattice parameters were found to be: a = 9.496 &#177;0.006 &#197;, b = 8.761 &#177;0.004 &#197;, c = 5.248 &#177;0.001 &#197;, &#946; = 105.06 &#177;0.03&#186;, &#945; = &#947; = 90&#186;.

In this report, we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell at the GSECARS 13-BM-C beamline at the Advanced Photon Source. ATREX and RSV programs are used to analyze the data.

高压单晶衍射PX ^ 2

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

用于大分子 X 射线晶体学的一体式样品支架,具有最小的背景散射

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

生物工程

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

We describe a beamline setup meant to carry out rapid two-dimensional x-ray fluorescence and x-ray microdiffraction mapping of single crystal or powder samples using either Laue (polychromatic radiation) or powder (monochromatic radiation) diffraction. The resulting maps give information about strain, orientation, phase distribution, and plastic deformation.

矿物和岩石样品的同步辐射 x 射线 Microdiffraction 和荧光成像

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder ...

工程学

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

酶的结晶和结构确定:多功能微流体芯片中串行晶体学的基板复合物

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

通过微透析对芯片上的蛋白质进行结晶，用于 原位 X射线衍射研究

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

室温下的片上结晶和大规模连续衍射

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

用于原位晶体 x 射线衍射和原位的微流控芯片系列晶体学的动态光散射

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