This work was supported by grants from the Deutsche Forschungsgemeinschaft to J.H. (FOR-1279, SFB-1078, B3). We thank Tom Resler and Bj&#246;rk S&#252;ss for helpful comments.

样品1。一般问题和仪器准备<ol><li>使用商用的FT-IR光谱仪装备时间分辨步进扫描测量。为获得最佳结果放置的FT-IR光谱仪上的振动解耦表的顶部。 </li><li>疏散的光学隔室到几毫巴。吹扫样品室与干燥空气或氮气。 </li><li>放置红外透明材料不透明的FT-IR光谱仪的MCT检测器的前面可见辐射。这种预防措施是必要的，以防止在步进扫描实验，从令人兴奋的激光脉冲文物。注意：我们使用锗直径25mm（GE）窗口。它的高折射率造成不良的强度损失，用葛滤波器抗反射涂层在很大程度上避免。 </li><li>降温MCT探测器杜瓦瓶液氮。注意：使用的光伏MCT检测器优于photoconductives因为它们的线性响应，因此，优于photometrIC的精度和可靠性的基准31,53。 </li><li>预浓缩用于实验，以至少2毫克蛋白/毫升的低的缓冲液（&lt;20毫摩尔）的离子强度，以防止形成盐晶体，同时干燥该蛋白悬浮液，形成均匀的膜蛋白质的样品。对于沉积物样品，洗/应用超速离心（ 例如 ，膜蛋白在脂质体或细胞膜补丁）集中起来。使用适当的截止centricons的蛋白质，不沉淀物（ 如洗涤剂溶解膜蛋白）。 </li><li>为获得最佳效果，请使用：1）蛋白制剂和纯粹的，活跃成为可能; B）脂/蛋白比值≤重量3在细胞膜补丁或再造在脂质体中的蛋白质;三）清洁剂/蛋白比值≤1的重量为洗涤剂溶解蛋白质。避免使用清洁剂或脂质，其振动谱带与蛋白质（ 例如 ，CHAPS）的重叠。 </li></oL&gt; 2， 细菌视紫红制备衰减全反射实验<ol><li>安装ATR附件进入FT-IR光谱仪的样品室。注意：我们使用的ZnSe /钻石ATR与5活跃的反射。避免半导体材料，例如锗或硅作为ATR附件的内反射元件（IRE），因为它们的光学性能受激磁可见激光。 </li><li>测量宽范围（约4,000-800厘米-1）能谱在4 cm-1处的清洁爱尔兰表面由传统的快速扫描傅立叶变换红外光谱仪的分辨率。使用此光谱作为参考光谱来计算吸收光谱在稍后的阶段。 </li><li>覆盖ATR的IRE表面与20μl后用再水合的样品（ 例如 ，4M NaCl和100mM的NAPI，在pH 7.4）的缓冲液中。测量缓冲器的IR吸收。 </li><li>除去缓冲液，并冲洗爱尔兰表面的Wi次水。避免接触表面。通过强烈的气流从爱尔兰表面去除残留的液体。 </li><li>在爱尔兰表面（约0.2 平方厘米面积）的顶部蔓延〜3微升细菌视紫红质（BR）在紫膜（〜6 mg蛋白/ ml的去离子水）。干燥该蛋白悬浮液下的干燥空气吹干直至膜达到。 </li><li>测定干燥膜的吸收光谱（ 见图1）。 </li><li>轻轻地添加20-40微升的高离子强度的再水合干燥的薄膜（ 例如 ，4M NaCl和100mM的NAPI，在pH 7.4）的缓冲液中。注意：高离子强度可防止膜膜的过度溶胀，允许保留尽可能多的蛋白质尽可能靠近被探测的表面（ 图2）。 </li><li>覆盖ATR架有盖，防止水分蒸发。任选地，将一个盖护套连接到循环浴设定为25℃的温度控制。对于长photocycles（＆＃蛋白质62，秒），温度控制可能会增加基线稳定性。 </li><li>测量的吸收光谱。估计样品的膜中减去缓冲液的吸收光谱（ 图3），再水化后残留的表面附近的百分比。 </li><li>确认影片通过记录吸收光谱在补液后5-20分钟的间隔（ 图4）肿胀的稳定。这一步是可选的，但建议。 </li></ol> 3，执行对洗涤剂溶解的Channelrhodopsin-2传输实验 <ol><li>加10微升的ChR2（〜10毫克蛋白质/毫升）溶解于5 mM氯化钠，5mM的HEPES（pH7.4）和癸基-麦芽糖苷（DM）在20mm直径的氟化钡窗口的中心。用微量移液器尖端的帮助下，以一个6-8毫米直径铺（〜200微克/平方厘米的蛋白质表面密度2）。 </li><li>形成薄膜USI纳克的干燥空气水流平缓。为了达到最佳效果，确保薄膜是均匀的，并且其直径在最大孔径大致匹配的红外光束的大小。 </li><li>通过将3-5微升甘油/水分布在3-5滴周围的干膜的混合物，并紧紧地贴着它与第二个窗口使用平面硅树脂O形圈的≥1毫米厚度水化膜。注意：在甘油/水混合物的比例控制窗54之间的相对湿度。对于吸湿性示例使用一个3/7或2/7甘油/水比（W / W）。的甘油/水滴不应该与蛋白质膜直接接触。 </li><li>插入支架的氟化钡夹窗口。从通过用红外线不透明的材料，如纸，有孔相匹配的水合的膜的尺寸覆盖了样本窗口到达检测器防止直光。将支架在样品室。 </li><li>控制保持的温度为25°下通过一个恒温夹套连接到一个温度控制的循环浴中。 </li><li>测定吸收光谱。保证在酰胺I区的最大吸收（〜1,700-1,620厘米-1）是在0.6-1.0的范围内。 </li><li>使用参考消光系数谱的水，DM，并代表的膜蛋白（ 图6）估计的质量和蛋白质/剂/水的摩尔比从水合的膜的吸收光谱（ 图5）。 </li><li>等到水化程度进行步进扫描实验（≥1小时）前稳定。 </li></ol> 4，调整了令人兴奋的激光和同步 对bR的实验使用脉冲Nd的二次谐波发射：YAG激光器（λ= 532纳米）来触发光周期。对于涉及的ChR2的实验中，用λ= 450nm的激发通过一个运算所提供tical参量振荡器（OPO）由一个钕的三次谐波（λ= 355纳米）：YAG激光器。确保激光脉冲足够短（〜10毫微秒），以最小化二次光激发。注意：激光脉冲的能量应尽可能保持恒定。在我们的设置中，激光强度波动≤10％左右的平均值，并经测量激光的反射由连接到一个瞬态记录器的光电二极管和集成的响应（ 图8）。 <ol><li>情侣激光在样品上。调整激光在样品上的能量密度，以使用功率计2-3毫焦耳/厘米2每脉冲。 <ol><li>在ATR设置，使用光纤放在头顶上的ATR盖的。 </li><li>用于传输的实验中，使用反射镜使激光在样品和如果需要，镜片要么准直或发散的激光束的直径稍微高于试样膜的大小。 </li></ol></li><li>落色同步r型光谱仪脉冲数据记录。使用Nd组成的电子器件的Q开关同步输出的TTL脉冲：YAG激光器来触发分光计。设置钕的激发率：YAG激光，以10赫兹的Br和0.25赫兹的ChR2，分别。 </li><li>使用一个脉冲延迟发生器（PDG）来控制激光激发速率，如果钕的重复率：YAG激光器是不容易调整的。 <ol><li>灯同步输出的钕连接：YAG激光对PDG外部触发输入。该PDG提供〜190微秒延迟的TTL脉冲输出到Q开关同步的钕输入：YAG激光器来触发激射。门的PDG接受外部触发后，才自最后一次接受的触发器（100毫秒Br或4秒，CHR 2）一定时间。 </li></ol></li></ol> 5，时间分辨步进扫描准备和设置<ol><li>放置一个低通滤光器在光路中。要执行在1,800-85时间分辨步进扫描测量0厘米-1的光谱范围内，可以使用一个滤波器不透明以上1950厘米-1，低于1800厘米-1（ 图7）良好的传输（&gt; 50-80％）。 </li><li>探测器从改变AC至DC耦合模式。注意：在此之后调整干涉信号将不再摆动围绕零但周围被称为DC-电平的值。 </li><li>使用分光计的最大可能光束孔径为高光子通量，因此，更好的信号 - 噪声，但在检测器的线性度的限制。 </li><li>把干涉图的DC电平为零，调整电子增益，以更好地利用模拟数字转换器（ADC）的动态范围。实现DC-电平通过施加电压偏压至由前置放大器提供的信号偏移。注意：此选项中包括现代的FT-IR光谱仪。否则，可以使用自制的外部设备来代替，放置在前置放大器和ADC 38之间。护理是那么需要确保这种设备的电子产品的快速和线性。 </li><li>启动的FT-IR光谱仪的步进扫描菜单。设置目标光谱带宽为步进扫描测量1,975.3-0厘米-1。调整频谱带宽的He-Ne激光的波数（在本例中的1/8 个 ）的一小部分，稍微超过该光学滤波器的截止。 </li><li>禁用任何高通电子滤波器，以防止动力学的扭曲在稍后的时间。一个低通电子滤波器可以是有益的，当其截止频率为顺序或以上的ADC（降低噪声混叠）的采样率。 </li><li>谱和相位分辨率设置为8 -1至64 -1，分别。前锋设置干涉采集模式为单端。有了这些选项之一干涉大约需要500点。 </li><li> ADC的采样速率设置为可用的最高的光谱仪（ 例如 ，160 KHz或6.25微秒）。设置“触发实验”到“外部”， 即激光是主人。设置要被记录的线性间隔的数据点的数目：7,000 BR（最多42毫秒）和20,000的ChR2（最多125毫秒）。注：较长的时间尺度可以不使用准对数时间间隔外部TTL脉冲从波发生器来触发数据采集38溢出ADC的内存被覆盖，或者使用两个平行瞬态记录作为内部ADC 31的替代品， 32。 </li><li>保存约100预触发点作为供试品的暗状态的参考。注意：在毫秒时间尺度的数据质量往往是在移动镜子波动有限。增加高于100的预触发点，因此，预计不会显著提高数据质量。 </li><li>设置共同增加的数量， 即每个镜像断定该光反应的平均次数离子。对于BR，使用20个合作增加（测量时间为10 Hz激励，税率为20分钟）。对于ChR2的使用2合加（测量时间为0.25赫兹激励率达70分钟） </li><li>重复实验10倍的Br和35倍三种不同的样品薄膜的ChR2，有终于〜200合作，每增加后视镜位置。 </li></ol> 6，数据处理<ol><li>通过比较由所述第一和最后一次测量得到的光致IR差异谱证实样品的状态。考虑丢弃其中的差异谱的强度下降到低于所述第一测量的60％的任何量测。 </li><li>平均记录的干涉。 <ol><li>使用从FTIR光谱仪的OPUS软件中选择的时间分辨干涉来平均，并将其添加到“频谱计算器”。 </li><li>点击“=”来获得干涉的平均值。 注意：当干涉图的相位是恒定的测量是淡漠平均干涉或单通道谱中。恒定的相位可以通过计算和相位谱的第一个和最后一个记录的干涉比较来证实。 </li></ol></li><li>进行平均化时间分辨干涉图的傅立叶变换，得到平均时间分辨单个信道的光谱。 <ol><li>使用相同的软件，如步骤6.2.1，选择平均时间分辨干涉，并点击“干涉到光谱”的图标。使用默茨相位校正方法，是2的零填充因数（每器乐分辨率两个数字点），并切趾函数（ 例如 ，三角形，Blackmann哈里斯-3 -条款等）。 </li><li>单击底部的“转换”，改造时间分辨干涉到时间分辨光谱。 </li></ol></li><li>导出的时间分辨单个信道的数据作为一个“数据点表”或“; matlab的“使用文件中的”另存为“，在OPUS软件图标。继续在一个外部程序的数据处理。 <ol><li>一般的单信道的光谱的激光之前，与时间分辨单个信道的数据转换成时间分辨吸收差谱。 </li><li>激光前的100个单信道的光谱的计算的预期噪声标准偏差的差示光谱作为波数（ 图12）的功能的载体，使用该噪声标准偏差，ε和平均，S 0（ν）： ε/ S 0（ν）。估计ε的值减去连续的单声道谱和计算√2校正标准偏差。 </li><li>删除光谱区域太吵被使用（ 例如 ，上述1825厘米-1，低于850 -1）。 </li><li>执行一个准对数求均（ 例如 ，20光谱/时间十年）。该appearan数据的CE将改善和光谱的数目会由10〜4降至〜10 2没有丢失任何显著信息（ 图9）。 </li><li>使用傅立叶插值点增加四倍的谱密度（以1点/厘米-1）（上传零填充）。 注：我们在执行在MATLAB中运行自制程序的步骤6.4.1至6.4.4。 </li></ol></li><li>处理所获得的时间分辨光谱（ 图10A）与奇异值分解，奇异值分解，（ 图13）。通过与显著SVD分量（ 图10B）有限数量的数据重建完成数据去噪。注意：我们在MATLAB中进行奇异值分解，使用内置的“SVD”功能。 </li></ol>

作者宣称没有竞争的经济利益。

一对蛋白质进行时间分辨步进扫描FT-IR实验时，需要考虑的第一个方面是一个示例的以适当的形式对红外光谱的制备。从较感兴趣的蛋白质等物质的红外吸收需要被降低，特别是，从水。最常见的方法是蒸发样品的体积的水，以形成薄膜。该薄膜可以通过添加的水溶液一些液滴或通过使该薄膜对受控湿度的气氛中进行再水化。我们已经展示了如何在这两种情况下，可以以预计的红外吸收光谱装置获得的水合水平（参见图3和图5）。虽然比起那些在溶液中所获得的水合水平可能会出现低，他们实际上接近于在活细胞中发现70，使得功能相关的蛋白质的水化膜的研究。除了水，它同样重要的是知道并控制脂质或洗涤剂样品中的量。既应保持足够低，以具有在红外吸收光谱降低的影响，但高到足以保持所感兴趣的蛋白的完整性和功能。 时间分辨步进扫描光谱是直截了当只适用于蛋白质显示，可以重复地利用光触发可逆反应（但看到进步的耦合步进扫描快速更换缓冲液）71。在步骤扫描的反应需要是可再现的，至少〜500倍，最小数目来完成的干涉覆盖1,800-850厘米-1区域8 -1的分辨率。然而在实践中，附加的数据平均化，通常需要与噪声电平向下推。 200 co-additions/mirror位置（10 5次重复反应）6.25微秒分辨率的吸收差光谱可以显示噪音站ndard偏差为2×10 -5到2×10 -4对的ATR和介于5×10 -6 -3×10 -5的传输实验（ 图15）。由于其较高的光子通量，传输允许〜7低噪音水平比ATR，成功地学习样本给弱的吸收变化，如ChR2的一个重要方面。另一方面，ATR需要5到25倍的传输实验样品量少。 时间分辨步进扫描红外光谱的应用是有问题的蛋白质显示缓慢photocycles：录制干涉一步扫描可以成为不切实际的长。有些解决方案已经提出了处理此类案件72,73，往往是基于使用多个交换样本，以加快测量在增加蛋白质消耗和实验的复杂27,74的成本。在某些情况下，可以由前绕过这个问题理由是样品前光周期是严格完成。对于ChR2的，具有光循环需要光激发60秒，99％恢复后，恢复为4秒已经是80％48。为每脉冲激光10％的激发效率，98％的ChR2分子是在黑暗状态下持续4秒的光激发后，使得能够在激光的重复频率，以0.25Hz的进行实验。 数据处理是为了达到最佳的效果需要一个最终的技术方面。对数平均减少了噪音，并且也很重要，利用奇异值分解或全局拟合减小数据的大小，而不扭曲，对后的数据分析功能是必不可少的。对数平均化，但是，不是在平均出在测量过程中的移动镜和其它的1 / f噪声源（ 图9）所引起的振荡的时间痕迹的波动非常成功。这些波动在基线可以超过在毫秒范围和数据损坏的质量的噪音。奇异值分解需要的数据，以减少噪声的冗余的优点，并且有一些修改57可减少以及在基线波动。 最后，时间分辨步进扫描FT-IR实验的困难和最耗时的部分对应于频带的分配和数据的光谱和动力学解释。对于许多细菌视紫红出现在红外差谱的乐队已分配或解释，由于许多研究者群体的几十年来积累的工作。对于少得多研究的蛋白质，如channelrhodopsin-2，上述介绍时间分辨红外实验需要伴随着对定点突变平行实验，并结合从互补技术的信息以达到机械的解释48。

到充分阐明蛋白质如何执行其功能时，需要对其进行测量，因为它们的工作， 即沿着往往涉及到一系列的中间体，可以延长时间几个数量级的反应路径。蛋白质功能的关键步骤往往发生在微秒到毫秒的时间范围1，特别是骨干网的构象变化和膜蛋白的质子转移反应。 X射线晶体学，结构生物学的可以说是支柱，提供了从井衍射蛋白质晶体静态的（时间平均）的电子密度，非常难以与膜蛋白2增长。可以根据电子密度建造一个三维的原子模型，虽然很少，包括氢原子的位置。在时间分辨X射线晶体学，无论是依托于劳厄衍射3或飞秒X射线脉冲4显着的进步，增加了时间维度，以在高结构信息相干到X-射线晶体5。但撇开技术和分析挑战，晶格能削弱骨干构象变化，并改变蛋白质动力学，方法基于蛋白质晶体5不可避免的缺点。因此，蛋白质动力学方面仍然是最好的覆盖光学方法，如率先通过闪光光解6,7，虽然与有限的结构有识之士的普遍缺点。 傅立叶变换红外光谱（FT-IR）结合光谱学的一个宝贵的敏感性蛋白质结构，利用在膜蛋白8-11无数的静态结构和功能研究的最后一个功能的时间分辨率。特别是，FT-IR光谱差异已被证明是研究极小的光谱变化作为蛋白质过境从一个亚稳状态到另一个12-19的理想工具。 科仪对蛋白质动力学，比在单分子水平20,21等，S需要进行同步的触发过程。反应便利地启动快，没有侵入性利用光作为触发，为已完成与循环光反应蛋白的研究。用时间分辨研究相关的一个主要挑战是实现足够的时间分辨率，同时保持良好的光谱分辨率和相应的信号噪声比。另外重要的是要涵盖了足够宽的光谱和时间范围。时间分辨步进扫描红外光谱擅长在所有这些方面22，与已公布的实例覆盖的光谱范围尽可能广泛的3,900-850厘米-1和动态扩展〜时间9目，以高达3.5 -1谱和30纳秒的时间分辨率23-28。 傅立叶变换红外光谱仪显示降低噪声和提高光度准确度分散的人29。 Howev尔，在正常快速扫描记录模式的FT-IR光谱仪遭受从时间分辨率限制为≥5-10毫秒为干涉仪的移动镜需要完成一个扫描的最小时间的结果。与步进扫描技术，与此相反，时间的动态事件的依赖性，从干涉仪的扫描持续时间解耦。简言之，在不连续的步骤，而不是该移动镜移动时连续扫描来完成扫描。在每个步骤的（移动）反光镜保持固定和瞬态记录。因此，时间分辨率由汞 - 镉 - 碲化物（MCT）检测器，它通常是在10-100毫微秒的范围内的上升时间的限制。在实践中，干涉图（包含在翼的centerburst和小信号的强烈信号）的大的动态范围，需要进行适当的数字化的模拟/数字转换器（ADC）与多达16-24位，导致取样率不大于〜200 kHz的更高（5微秒）30。纳秒时间分辨率可以通过测量只改变干涉，为此，一8-12位ADC是足够23,31-33来完成。技术方面30,34,35和应用时间分辨步进扫描的36-38已经详细地讨论过别处。 的当前贡献的目的是提供描述的时间分辨步进扫描的FT-IR光谱仪上的光敏膜蛋白的实用性的协议。这里，该技术的性能示于2取样方法：变速器和衰减全反射（ATR）。使用ATR允许在过量水存在，这不仅保证了全水化条件的蛋白质，但也允许样品的pH值和离子强度49,50急性控制工作。步进扫描实验说明在两个选定的系统​​：细菌视紫红质和channelrhodopsin-2。 39,40，使其成为最了解膜蛋白为止。其中适用于bR的功能FT-IR光谱研究中的许多技术已经可以说是发挥的最大影响之一。也就是说，FT-IR光谱是关键，以解决涉及跨膜质子转移组与其他地方一样13,41,51核算。 Channelrhodopsin（CHR）是在自然界中发现42,43的第一光门控离子通道。 CHR的光激发导致的离子通道的瞬时开口。它的发现为光遗传学，其中分子过程是由光44,45控制的入驻发展的方式。 CHR所属，如BR，微生物的视紫质的家庭，但在对比BR，不太有人知道它的作用机制52。的ChR2结合其功能作为ION沟道与质子泵活性46,47。最近，我们采用时间分辨步进扫描红外光谱来解决同蛋白质质子转移反应，并在ChR2的48蛋白质主链构象变化的动态。

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			<td>Equipped with photovoltaic-MCT detector, an external global, and an oil-free pump. Firmware 2.3.</td>
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			<td height="21" style="height:21px;width:216px;">OPUS 7.0&#160;</td>
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			<td>Software to control Vertex 80v spectrometer</td>
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			<td>Used to run home-made executable programs to preprocess the data</td>
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proton transfer and protein conformation dynamics in photosensitive proteins by time-resolved step-scan fourier-transform infrared spectroscopy

监测的质子化和蛋白骨架的构象变化的动态的蛋白质的功能时是理解其机制的重要一步。质子化和构象变化的影响的氨基酸侧链与肽键，振动模式分别，这两者都可以通过红外（IR）光谱的差异来探测。蛋白质，其功能可以是反复地和可重复性触发灯，它有可能获得的红外光谱差异与在很宽的光谱范围内使用步进扫描傅立叶变换红外光谱法（分）微秒的分辨率。用〜10 2 -10 3次重复的光反应，最小数目来完成以合理的光谱分辨率和带宽的扫描，在吸收差光谱的噪声电平可以是低至〜10 - 4，足以跟随的动力学从单个氨基酸的质子化的变化。下噪声电平可以由多个数据平均化和/或数学处理来实现。所需的最佳结果蛋白质的量为5-100微克之间，取决于所使用的抽样技术。对于额外的要求，该蛋白质必须首先集中在低离子强度缓冲液，然后干燥以形成薄膜。该蛋白质膜是在实验之前，水合，无论是与水的小液滴或在受控的大​​气湿度。所达到的水合水平（克水/克蛋白质）是由IR吸收光谱衡量。为了展示该技术，我们研究了光驱动质子泵细菌视紫红质在其原生紫膜环境的光周期，和光门控离子通道channelrhodopsin-2溶解洗涤剂。

图1示出bR的沉积用于ATR光谱的钻石内反射元件的表面上的干燥膜的吸收光谱。从肽键（酰胺一，酰胺Ⅰ和酰胺Ⅱ）的振动特征峰都清晰可辨。干膜的厚度约为可估计为〜1微米，考虑到附加的蛋白（18微克）的量和ATR的表面（约0.2 平方厘米），并考虑蛋白质的浓度为〜1.4克/厘米3，58脂质和1.0克/厘米3，59和1/3（重量/重量）的紫膜60的脂质/蛋白质比。 的干燥膜再水化用过量的4M的NaCl，100mM的NAPI在pH为7.4（ 图3）。在由倏逝波探测的量的水合程度，有效的蛋白质浓度可以从所需的缩放因子推导出以数字方式去除吸收带从水。最佳比例因子为0.87，这意味着在这种情况下，缓冲器占有87％，表面附近的该卷的样品的13％。考虑到蛋白质和脂质的密度和在紫膜的脂质/蛋白质比（见上文），我们可以推断出的125 mg / ml的一种有效的蛋白浓度在由倏逝波探测的体积。我们可以推断从水化水平，在这种特定情况下，该样品的膜厚时再水化膨胀〜6倍，从约1至约6微米。为45°的入射角，典型为和为最ATR安排，水合蛋白膜的穿透深度的渐逝场（D P）的0.3至0.6微米的1,800-850 -1间隔61而变化。因为渐逝场几乎是不敏感的位于2倍以上D P 61的示例中，我们推断，在ATR实验中使用的蛋白质的量可以是在原理降低5倍瓦特ithout任何显著信号丢失。 当使用低离子强度缓冲液的膜，越来越多，以及蛋白质在由渐逝场探测的体积量被减少（ 图2）。膜溶胀和缓冲液的离子强度之间的精确的依赖将取决于其他因素，对脂质的性质。例如，被用于在重组大肠杆菌极性膜蛋白获得类似电影中使用4 M氯化钠紫膜在这里得到对于BR肿胀只用0.1 M氯化钠62 大肠杆菌脂质。如果样本占用探测体积小于5％考虑使用较高离子强度的缓冲液重新做的水合步骤。薄膜水合后肿胀，需要一些时间来达到稳定（ 图4）。对于BR在紫膜的过程是单指数，用12分钟的时间常数：花费不超过30-60分钟，有一个稳定的水化膜<p级=“jove_content”&gt; 图5示出的ChR2的由传输获得的水合薄膜的IR吸收光谱。这里，水化是通过将干膜以控制湿度的由甘油/水的混合物中提供的气氛中实现。的频谱可以被分解成贡献水，洗涤剂和蛋白。我们可以预计使用消光系数光谱他们每个人的量，缩放，以适应实验谱。对于水，我们采取了消光系数的频谱从文献63，以及糖尿病，我们测量了从100毫克/毫升的溶液（ 图6）。我们还使用消光系数为2代表的膜蛋白：线粒体ADP / ATP载体64和BR（ 图7）。缩放因子表示为260微克/厘米2和200微克/厘米2的膜的水和DM存储表面密度。其余的吸收（FIGUR以东5，红线）主要来源于蛋白质，估计是在250微克/厘米2使用酰胺Ⅱ消光系数从两个不同的膜蛋白的表面密度（ 见图6）。的摩尔比为蛋白质/剂/水的计算使用35 kDa的，480道尔顿，和18沓，分别的分子质量为1/60/2000。 3D绘图与典型的时间分辨步进扫描红外光谱实验研究BR是显示在图10A，由ATR获得，涉及200分钟数据采集。光谱可以在特定的时间提取出来，例如当L时，对BR光循环的M和N的中间体有望达到其最高的人口（ 图11）。它们的光谱特征已被广泛13,41,65描述，将不会进一步讨论在这里。 图12显示时间的痕迹在一些选定的波数。也就是说，在1,762 c中的动力学崛起米-1（ 吨 1/2〜60微秒）报告了Asp85质子化的视网膜Schiff碱66的动力，并且其衰减到零表示其在地面沙爹恢复67去质子化。动力学在1740厘米-1（叔1/2〜1毫秒）的负上升汇报Asp96侧链的质子供体的Schiff碱68的去质子化。的强度，以对从细胞质67其质子化零报告的衰变。的蛋白的构象变化的动力学可以通过在酰胺I和酰胺Ⅱ区域，达到在约3毫秒室温67,69的最大变化吸收的变化来探测。 SVD的应用，光谱问题55,56之前进行了审查。简单地说，SVD factorizes实验数据，排列成矩阵A，因为A =美 <stroNG&gt; V T。这种分解可以进行修改，以考虑对波数的噪声依赖和惩罚波动/漂移基线57。 U和 V的列中包含正交光谱和时间轨迹矢量对每个SVD分量。S是含有所谓的奇异值的对角矩阵。在U和 V的（抽象）分光时间分量出现在下降关联来描述在最小二乘意义上，通过它们相关的奇异值量化的实验数据。 图13A示出的奇异值作为组分数量的函数，并且再现U（抽象的光谱， 图13B）和V（抽象的时间痕迹， 图13C）的前八列/组件。该信号集中在第一个五年组件（如预期的pH值otocycle包含五个中间状态），与上述部件由随机噪声和错误的其他来源主要是占主导地位。仅使用前五个栏U和 V，而前五列和行S的实验数据进行重建。通过SVD重建中的数据表示实验数据的秩五个矩阵的最佳最小二乘逼近。重建的数据显示改进的质量（ 图10B）。即，噪声被大大降低，以及某些几乎没有明显的波动和漂移的基线（在时间分辨步进扫描光谱仪25通用）。 SVD的处理是用于提高试验时的痕迹在毫秒范围内（ 图12中的红色线）的质量特别有益。 采用上述协议，用于透射，获得时间分辨步进扫描数据为ChR2的光循环<html>锡永的实验（ 图14A），大约涉及120小时累积测量。由步进扫描收集的时间分辨数据的范围是从6.25微秒至125毫秒。的ChR2的光周期大约需要60秒的全面复苏。在光周期的最新部分可以使用快速扫描FT-IR被覆盖，并且两个步进扫描，快速扫描数据集的合并与其他地方一样呈现48。的ChR2的光谱变化〜高于10倍小获得BR，使得测量数据更​​有挑战性。特别地，振荡在基准在毫秒范围变得显而易见在这些低的吸收的变化。这是由于在毫秒时间尺度25移动镜子小的振荡。的振荡，与噪声的部分一起，可以在很大程度上由奇异值分解去除，显着地提高了数据的外观（ 图14B）。 GURE 1“FO：内容宽度=”5英寸“SRC =”/ files/ftp_upload/51622/51622fig1highres.jpg“宽度=”500“/&gt; bR的在紫膜如图1所示。吸收光谱上干燥ATR附件的顶金刚石表面。由肽键的振动带（酰胺I，II，和A）表示。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig2highres.jpg" width="500" /> bR的再水化后使用各种离子强度的缓冲液的膜的图2。吸收光谱（4M的氯化钠，100毫米的Na 2 HPO 4的稀释/加入NaH 2 PO 4，在pH 7.4）中，注意酰胺II带的减小， 也就是说 ，增加了电影肿胀，与减少缓冲区的离子强度。 （插入）相对于蛋白质的量的表面附近，在1541厘米-1（酰胺Ⅱ最大）subtracti后定量的吸收的强度上的缓冲器的吸收的贡献。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig3highres.jpg" width="500" /> bR的再水化的散装缓冲液（430万的离子强度）和缓冲贡献减法后的膜的图3。吸收光谱的加减系数，0.87，被选择去除的强吸水性3,700-3,000厘米-1之间以及获得2,700-1,800厘米-1之间平坦的基线。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig4highres.jpg" width="500" /> bR的补液为430万的离子强度的缓冲液后，如图4所示。吸收光谱（吸收缓冲器已经减去为了清楚起见，如图3），插入件中给出了膜rehydrat后肿胀的演变离子，其次是蛋白质酰胺Ⅱ吸光度1541厘米-1。一件合身的单指数表示时间常数的12分钟电影肿胀稳定。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig5highres.jpg" width="500" /> 的ChR2的由传输（蓝线）测得的水合的膜的图5。吸收光谱。水的消光系数谱（虚线橙色线）和DM（虚线青色线）中的比例和相减（红线）。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig6highres.jpg" width="500" /> 在25℃下为液态的水（http://www.ualberta.ca/〜jbertie / JBDownload.HTM＃光谱）和用于ADP / ATP载体（AAC）的水合膜图6。转载质量消光系数谱64 。 </s<html> TRONG&gt;质量消光系数谱：在对德国马克（100毫克/毫升）的解决方案，并在对于BR水化膜。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig7highres.jpg" width="500" /> 图7。透过率在时间分辨扫描扫描的FT-IR测量中使用的滤光器的。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig8highres.jpg" width="500" /> 由钕的第二谐波（532nm），提供了激光脉冲的图8中的性能：1,000激光脉冲的相对能量变化的YAG激光的直方图，并适合于高斯分布具有0.05的标准偏差。 1622/51622fig9highres.jpg“宽度=”500“/&gt; 图9。光致bR的吸光度变化在在统一的时间三个有代表性的波数间隔的时间间隔（绿，黑，蓝线）和半对数平均后〜20点/十年（红色线），注意对降噪后的对数平均，揭示了时间的振荡轨迹在毫秒范围内。 <img alt="图10" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig10highres.jpg" width="500" /> 在pH 7.4（4 M氯化钠，100毫米NAPI）录得ATR对于BR光致吸光度的变化图10。3D表示。 A）原始数据B）重建与五SVD组件的数据。 <a href="https://www.jove.com/files/ftp_upload/51622/51622fig10highres.jpg" target="_blank">请点击此处查看此图的放大版本。</a> 图11。对BR光循环的三个选定的时间，其中L（12.5微秒），M（300微秒）和N（6毫秒）中间体的最丰富的FT-IR差异吸收光谱。 <img alt="图12" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig12highres.jpg" width="500" /> 为解决由时间分辨步进扫描红外差谱在BR光循环图12。质子转移和蛋白质骨架动力学。在1762厘米-1 Asp85的质子化/去质子化的动态报告，并在1741厘米的吸光度变化-1 Asp96的脱质子/质子化动力学（包括前300微秒氢键的变化）。在时间的痕迹在1,670和1555厘米-1在酰胺I报告的变化和II的振动，无论是敏感的肽主链的构象。背部走线对应的原始数据，并SVD处理过的数据红色的。 <a href="https://www.jove.com/files/ftp_upload/51622/51622fig12highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图13" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig13highres.jpg" width="500" /> 图13。对BR光循环的实验时间分辨步进扫描红外光谱数据的奇异值分解（SVD）（见图10A）。SVD称重的实验数据时，应考虑波数的噪声标准差的依赖后进行（ 图15）;与第一导数结合，以减少基线波动的统计权重，如前57。A所述）＆＃160;剧情第50组件（黑圈）的相对奇异值。前五个元件被分配到的信号成分（红色圆圈）。组件衰减指数如预期的噪声成分的剩余部分（见灰色虚线），B）前八的抽象光谱（U 1至U 8）C）的前八个抽象的时间痕迹（V 1至V 8）。抽象的光谱和时间的痕迹分配到的信号中描绘了红色线，黑线，否则， <a href="https://www.jove.com/files/ftp_upload/51622/51622fig13highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图14" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig14highres.jpg" width="500" /> 为ChR2的记录步进扫描光致红外吸收变化图14。3D表示在传输模式下的步进扫描数据扩展到125毫秒，只覆盖了光周期A）的一部分原始数据B）重建与五SVD组件的数据。 <a href="https://www.jove.com/files/ftp_upload/51622/51622fig14highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图15" fo:content-width="5in" src="/files/ftp_upload/51622/51622fig15highres.jpg" width="500" /> 中的FT-IR。差分吸收光谱在图15中。估计噪声电平 6.25微秒的时间和8 -1，涉及1 co-addition/mirror位置（500光反应）或〜200 co-addition/mirror位置的实验光谱分辨率（10 5光反应）。值显示为衰减全反射（ATR）和传输设置。

Watch this Scientific Journal Video about Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy at JoVE.com

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

质子转移和蛋白质构象动力学的光敏蛋白的时间分辨步进扫描傅立叶变换红外光谱

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards ...

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17.8K 次观看.  Freie Universität Berlin. Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

视频: Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

调查单分子粘附的原子力谱

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

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Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

检测和钯的回收，从城市矿山黄金和钴金属使用标有纳米货车车轮形的孔隙新型传感器/吸附剂

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Integration of Light Trapping Silver Nanostructures in Hydrogenated Microcrystalline Silicon Solar Cells by Transfer Printing

One of the potential applications of metal nanostructures is light trapping in solar cells, where unique optical properties of nanosized metals, commonly known as plasmonic effects, play an important role. Research in this field has, however, been impeded owing to the difficulty of fabricating devices containing the desired functional metal nanostructures. In order to provide a viable strategy to this issue, we herein show a transfer printing-based approach that allows the quick and low-cost integration of designed metal nanostructures with a variety of device architectures, including solar cells. Nanopillar poly(dimethylsiloxane) (PDMS) stamps were fabricated from a commercially available nanohole plastic film as a master mold. On this nanopatterned PDMS stamps, Ag films were deposited, which were then transfer-printed onto block copolymer (binding layer)-coated hydrogenated microcrystalline Si (&#181;c-Si:H) surface to afford ordered Ag nanodisk structures. It was confirmed that the resulting Ag nanodisk-incorporated &#181;c-Si:H solar cells show higher performances compared to a cell without the transfer-printed Ag nanodisks, thanks to plasmonic light trapping effect derived from the Ag nanodisks. Because of the simplicity and versatility, further device application would also be feasible thorough this approach.

A viable transfer printing-based methodology to introduce plasmonic metal nanostructures in solar cells is described. Using nanopillar poly(dimethylsiloxane) stamps, an Ag-based ordered nanodisk array was integrated with standard hydrogenated microcrystalline Si solar cells, which led to improved device performances due to plasmonic light trapping.

灯光诱杀银纳米结构通过转移印花在氢化微晶硅太阳能电池集成

One of the potential applications of metal nanostructures is light trapping in solar cells, where unique optical properties of nanosized metals, commonly ...

Characterizing Far-infrared Laser Emissions and the Measurement of Their Frequencies

The generation and subsequent measurement of far-infrared radiation has found numerous applications in high-resolution spectroscopy, radio astronomy, and Terahertz imaging. For about 45 years, the generation of coherent, far-infrared radiation has been accomplished using the optically pumped molecular laser. Once far-infrared laser radiation is detected, the frequencies of these laser emissions are measured using a three-laser heterodyne technique. With this technique, the unknown frequency from the optically pumped molecular laser is mixed with the difference frequency between two stabilized, infrared reference frequencies. These reference frequencies are generated by independent carbon dioxide lasers, each stabilized using the fluorescence signal from an external, low pressure reference cell. The resulting beat between the known and unknown laser frequencies is monitored by a metal-insulator-metal point contact diode detector whose output is observed on a spectrum analyzer. The beat frequency between these laser emissions is subsequently measured and combined with the known reference frequencies to extrapolate the unknown far-infrared laser frequency. The resulting one-sigma fractional uncertainty for laser frequencies measured with this technique is &#177; 5 parts in 107. Accurately determining the frequency of far-infrared laser emissions is critical as they are often used as a reference for other measurements, as in the high-resolution spectroscopic investigations of free radicals using laser magnetic resonance. As part of this investigation, difluoromethane, CH2F2, was used as the far-infrared laser medium. In all, eight far-infrared laser frequencies were measured for the first time with frequencies ranging from 0.359 to 1.273 THz. Three of these laser emissions were discovered during this investigation and are reported with their optimal operating pressure, polarization with respect to the CO2 pump laser, and strength.

We describe the generation of far-infrared radiation using an optically pumped molecular laser along with the measurement of their frequencies with heterodyne techniques. The experimental system and techniques are demonstrated using difluoromethane (CH2F2) as the laser medium whose results include three new laser emissions and eight measured laser frequencies.

表征远红外激光辐射及其频率的测量

The generation and subsequent measurement of far-infrared radiation has found numerous applications in high-resolution spectroscopy, radio astronomy, and ...

Emission Spectroscopic Boundary Layer Investigation during Ablative Material Testing in Plasmatron

Ablative Thermal Protection Systems (TPS) allowed the first humans to safely return to Earth from the moon and are still considered as the only solution for future high-speed reentry missions. But despite the advancements made since Apollo, heat flux prediction remains an imperfect science and engineers resort to safety factors to determine the TPS thickness. This goes at the expense of embarked payload, hampering, for example, sample return missions. 
Ground testing in plasma wind-tunnels is currently the only affordable possibility for both material qualification and validation of material response codes. The subsonic 1.2MW Inductively Coupled Plasmatron facility at the von Karman Institute for Fluid Dynamics is able to reproduce a wide range of reentry environments. This protocol describes a procedure for the study of the gas/surface interaction on ablative materials in high enthalpy flows and presents sample results of a non-pyrolyzing, ablating carbon fiber precursor. With this publication, the authors envisage the definition of a standard procedure, facilitating comparison with other laboratories and contributing to ongoing efforts to improve heat shield reliability and reduce design uncertainties.
The described core techniques are non-intrusive methods to track the material recession with a high-speed camera along with the chemistry in the reactive boundary layer, probed by emission spectroscopy. Although optical emission spectroscopy is limited to line-of-sight measurements and is further constrained to electronically excited atoms and molecules, its simplicity and broad applicability still make it the technique of choice for analysis of the reactive boundary layer. Recession of the ablating sample further requires that the distance of the measurement location with respect to the surface is known at all times during the experiment. Calibration of the optical system of the applied three spectrometers allowed quantitative comparison. At the fiber scale, results from a post-test microscopy analysis are presented.

Development of new ablative materials and their numerical modeling requires extensive experimental investigation. This protocol describes procedures for material response characterization in plasma flows with the core techniques being non-intrusive methods to track the material recession along with the chemistry in the reactive boundary layer by emission spectroscopy.

烧蚀材料在试验过程中等离子体发生器发射光谱边界层研究

Ablative Thermal Protection Systems (TPS) allowed the first humans to safely return to Earth from the moon and are still considered as the only solution ...

Free-form Light Actuators &#8212; Fabrication and Control of Actuation in Microscopic Scale

Liquid crystalline elastomers (LCEs) are smart materials capable of reversible shape-change in response to external stimuli, and have attracted researchers&#39; attention in many fields. Most of the studies focused on macroscopic LCE structures (films, fibers) and their miniaturization is still in its infancy. Recently developed lithography techniques, e.g., mask exposure and replica molding, only allow for creating 2D structures on LCE thin films. Direct laser writing (DLW) opens access to truly 3D fabrication in the microscopic scale. However, controlling the actuation topology and dynamics at the same length scale remains a challenge.
In this paper we report on a method to control the liquid crystal (LC) molecular alignment in the LCE microstructures of arbitrary three-dimensional shape. This was made possible by a combination of direct laser writing for both the LCE structures as well as for micrograting patterns inducing local LC alignment. Several types of grating patterns were used to introduce different LC alignments, which can be subsequently patterned into the LCE structures. This protocol allows one to obtain LCE microstructures with engineered alignments able to perform multiple opto-mechanical actuation, thus being capable of multiple functionalities. Applications can be foreseen in the fields of tunable photonics, micro-robotics, lab-on-chip technology and others.

Free-form Light Actuators &amp;#8212; Fabrication and Control of Actuation in Microscopic Scale

Here, we fabricate 3D polymeric micro/nano structures in which both the shape and the molecular alignment can be engineered with nanometer scale accuracy by the use of direct laser writing. Light induced deformation of several types of liquid crystalline elastomer microstructures can be controlled in the microscopic scale.

自由形式的光驱动器 - 制作和开动控制在微观尺度

Liquid crystalline elastomers (LCEs) are smart materials capable of reversible shape-change in response to external stimuli, and have attracted ...

The Evolution of Silica Nanoparticle-polyester Coatings on Surfaces Exposed to Sunlight

Corrosion of metallic surfaces is prevalent in the environment and is of great concern in many areas, including the military, transport, aviation, building and food industries, amongst others. Polyester and coatings containing both polyester and silica nanoparticles (SiO2NPs) have been widely used to protect steel substrata from corrosion. In this study, we utilized X-ray photoelectron spectroscopy, attenuated total reflection infrared micro-spectroscopy, water contact angle measurements, optical profiling and atomic force microscopy to provide an insight into how exposure to sunlight can cause changes in the micro- and nanoscale integrity of the coatings. No significant change in surface micro-topography was detected using optical profilometry, however, statistically significant nanoscale changes to the surface were detected using atomic force microscopy. Analysis of the X-ray photoelectron spectroscopy and attenuated total reflection infrared micro-spectroscopy data revealed that degradation of the ester groups had occurred through exposure to ultraviolet light to form COO&#903;, -H2C&#903;, -O&#903;, -CO&#903; radicals. During the degradation process, CO and CO2 were also produced.

Two types of surfaces, polyester-coated steel and polyester coated with a layer of silica nanoparticles, were studied. Both surfaces were exposed to sunlight, which was found to cause substantial changes in the chemistry and nanoscale topography of the surface.

表面上的纳米硅聚酯涂料暴露在阳光下的演变

Corrosion of metallic surfaces is prevalent in the environment and is of great concern in many areas, including the military, transport, aviation, ...

Recombination Dynamics in Thin-film Photovoltaic Materials via Time-resolved Microwave Conductivity

A method for investigating recombination dynamics of photo-induced charge carriers in thin film semiconductors, specifically in photovoltaic materials such as organo-lead halide perovskites is presented. The perovskite film thickness and absorption coefficient are initially characterized by profilometry and UV-VIS absorption spectroscopy. Calibration of both laser power and cavity sensitivity is described in detail. A protocol for performing Flash-photolysis Time Resolved Microwave Conductivity (TRMC) experiments, a non-contact method of determining the conductivity of a material, is presented. A process for identifying the real and imaginary components of the complex conductivity by performing TRMC as a function of microwave frequency is given. Charge carrier dynamics are determined under different excitation regimes (including both power and wavelength). Techniques for distinguishing between direct and trap-mediated decay processes are presented and discussed. Results are modelled and interpreted with reference to a general kinetic model of photoinduced charge carriers in a semiconductor. The techniques described are applicable to a wide range of optoelectronic materials, including organic and inorganic photovoltaic materials, nanoparticles, and conducting/semiconducting thin films.

A Time Resolved Microwave Conductivity technique for investigating direct and trap-mediated recombination dynamics and determining carrier mobilities of thin film semiconductors is presented here.

通过时间分辨微波电导率重组动力学薄膜光伏材料

A method for investigating recombination dynamics of photo-induced charge carriers in thin film semiconductors, specifically in photovoltaic materials ...

Measurements of Soil Carbon by Neutron-Gamma Analysis in Static and Scanning Modes

The herein described application of the inelastic neutron scattering (INS) method for soil carbon analysis is based on the registration and analysis of gamma rays created when neutrons interact with soil elements. The main parts of the INS system are a pulsed neutron generator, NaI(Tl) gamma detectors, split electronics to separate gamma spectra due to INS and thermo-neutron capture (TNC) processes, and software for gamma spectra acquisition and data processing. This method has several advantages over other methods in that it is a non-destructive in situ method that measures the average carbon content in large soil volumes, is negligibly impacted by local sharp changes in soil carbon, and can be used in stationary or scanning modes. The result of the INS method is the carbon content from a site with a footprint of ~2.5 - 3 m2 in the stationary regime, or the average carbon content of the traversed area in the scanning regime. The measurement range of the current INS system is &#62;1.5 carbon weight % (standard deviation &#177; 0.3 w%) in the upper 10 cm soil layer for a 1 hmeasurement.

Here, we present the protocol for in situ measurement of soil carbon using the neutron-gamma technique for single point measurements (static mode) or field averages (scanning mode). We also describe system construction and elaborate data treatment procedures.

静态和扫描模式下中子伽马分析法测量土壤碳

The herein described application of the inelastic neutron scattering (INS) method for soil carbon analysis is based on the registration and analysis of ...

Quantitative Analysis of Vacuum Induction Melting by Laser-induced Breakdown Spectroscopy

Vacuum induction melting is a popular method for refining high purity metal and alloys. Traditionally, standard process control in metallurgy involves several steps, include drawing samples, cooling, cutting, transport to the laboratory, and analysis. The whole analysis process requires more than 30 minutes, which hinders on-line process control. Laser-induced breakdown spectroscopy is an excellent on-line analysis method that can satisfy the requirements of vacuum induction melting because it is fast and noncontact and does not require sample preparation. The experimental facility uses a lamp-pumped Q-switched laser to ablate melted liquid steel with an output energy of 80 mJ, a frequency of 5 Hz, a FWHM pulse width of 20 ns, and a working wavelength of 1,064 nm. A multi-channel linear charge coupled device (CCD) spectrometer is used to measure the emission spectrum in real time, with a spectral range from 190 to 600 nm and a resolution of 0.06 nm at a wavelength of 200 nm. The protocol includes several steps: standard alloy sample preparation and an ingredient test, smelting of standard samples and determination of the laser breakdown spectrum, and construction of the elements concentration quantitative analysis curve of each element. To realize the concentration analysis of unknown samples, the spectrum of a sample also needs to be measured and disposed with the same process. The composition of all main elements in the melted alloy can be quantitatively analyzed with an internal standard method. The calibration curve shows that the limit of detection of most metal elements ranges from 20-250 ppm. The concentration of elements, such as Ti, Mo, Nb, V, and Cu, can be lower than 100 ppm, and the concentrations of Cr, Al, Co, Fe, Mn, C, and Si range from 100-200 ppm. The R2 of some calibration curves can exceed 0.94.

During vacuum induction melting, laser-induced breakdown spectroscopy is used to perform real-time quantitative analysis of the main-ingredient elements of a molten alloy.

激光击穿光谱对真空感应熔融的定量分析

Vacuum induction melting is a popular method for refining high purity metal and alloys. Traditionally, standard process control in metallurgy involves ...