Name: high resolution phonon-assisted quasi-resonance fluorescence spectroscopy
Uploaded: 2016-06-28T13:00:00.000+00:00
Duration: 10 min 40 s
Description: Watch this Scientific Journal Video about High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy at JoVE.com

作者要感谢艾伦·布雷克和丹尼尔金门海军研究实验室提供所研究的样本。这项工作是由美国国防威胁降低局，基础研究奖＃HDTRA1-15-1-0011，到加州大学默塞德分校（部分）的支持。

注意:所述的方法是针对特定的软件，虽然其他软件包可以替代使用。 1.样品制备和降温<ol><li>制作样品。 <ol><li>生长的样品，通过分子束外延生成由一个4纳米隧道势垒如前所述分开的两个垂直堆叠的自组装的InAs /砷化镓量子点使用斯特兰斯基-Krastanov生长方法16嵌入量子点中的电场效应结构（ 即 ，肖特基二极管）允许的电场被施加到QDMS 17 注意:使用QDMS的不是该方法的要求。另外，半导体的InAs /砷化镓是没有必要的，该技术将用于从任何半导体组合制成QDMS或量子点工作。 </li><li>制造使个别的量子点可以被光学寻址的样品。通过添加光圈面具样品或M顶做到这一点亚庆用10 8量子点/ cm 2以下取决于焦斑大小低密度样品18 </li></ol></li><li>装入样品到芯片标题。 <ol><li>敷在陶瓷芯片头由50％的铋，26.7％的铅，13.3％的锡和10％的镉的合金。使用热板，直到合金液化加热芯片。放置样品的底部到所述液化的合金其连接到芯片标题。 注:样品的底部是被连接到销的肖特基二极管和焊料点到芯片头的电极中的一个。用于连接样品的另一个备用的导电银环氧树脂。 </li></ol></li><li>坚持从点（上角）样品40庚金线连接到芯片上的销。 <ol><li>放置银环氧树脂的液滴在样品的顶部角落和在芯片上的销焊盘之一一个液滴。 </li><li>仔细躺在两个液滴的金线。 注:T的顶部他样品是肖特基二极管，其允许一个施加电场的另一个电极。 </li></ol></li><li>安装芯片和样品放入低温恒温器和确保样品具有与铜样品夹持器良好的热接触。 <ol><li>应用芯片和低温恒温器的冷指之间的铟箔。 </li><li>压力的芯片安装到冷的手指。用两个带垫圈螺丝拧紧牢固，以确保与低温恒温器的铜冷指良好的热接触。 </li></ol></li><li>从连接到既在样品的顶部和底部电极的芯片引脚连接电线。通过低温恒温器的源米跑这些电线。 注意:源计施加偏压到样品的电极时，量子点暴露于外部电场。 </li><li>疏散和使低温恒温器和样品室至真空。启动涡轮泵，抽真空至10 -6托，准备凉SAM下来PLE。 注:冷却和温度控制为实验用它由一个封闭的循环冰箱和一个附加的显微样品室的低温恒温器来实现的。 </li><li>启动低温恒温器的压缩机。允许低温恒温器冷却系统，直至达到所需的温度。 注意:对于提出的结果，温度为大约18 K。一旦样品冷却，设置准备用于安装光学器件，这将允许光学测量将要采取的。 </li></ol> 2.光学设置注意:对于所有的设定程序，使用由制造商或其他自定义程序提供的软件通过任何运行激光，光源仪，光谱仪和CCD。 <ol><li>用于PL收集，放置一个长工作距离50X显微镜物镜和准直透镜符合的PL信号聚焦到光谱仪镜头。通过0.75毫米收集频谱onochromator其中信号由1100毫米-1光栅分散并使用液氮检测冷却1340×100像素的CCD照相机。 </li><li>使用白色光源，照射样品。 <ol><li>通过适当地对准在检测侧的所有光学元件（ 即，准直和聚焦透镜），并在零波长获得分光计CCD样品的清洁聚焦图像集中样品的图像通过外部照相机和分光计。 注意:这是有帮助的另一外部成像相机，以帮助对准和获取样本的清晰的图像。 </li></ol></li><li>检测侧建立后，聚焦激光到样品。通过使用透镜聚焦光束点到样品上的最小尺寸成为可能。使用可调谐二极管激光器与包含基态跃迁能量的激励范围。以斜角设置激光入射到样品中。斜入射的一个优点是它有助于摆脱散射的激光的很大一部分。 </li><li>激发在更高的非谐振能量的样本。理想情况下，在润湿层之下的能量做到这一点通过激励。对于砷化铟/砷化镓QDMS在这项研究中，这相当于75兆电子伏以上的高于地面状态转换。 </li><li>运行对焦模式光谱采集软件。扫描整个使用被连接到低温恒温器镜样品壳体的XY平移台激光点的样品。这样做，直到光谱仪CCD捕捉到基态跃迁的离散线。正中央的QDMS之一的检测。 注意:一旦QDM发现，光安装完成。对于采用基态能量的样品约1300毫电子伏特。 </li><li>产生偏见地图。 <ol><li>申请经由连接到样品上的电极上的源表（步骤1.5）的电位;这适用反过来在电极的偏压产生的电场向QDM秒。 注:施加于样品的偏差范围是0-2伏穿过肖特基二极管结构。这是当该装置处于反向偏置，并且电场限制在QDM允许各个充电状态是可见的电荷量。 </li><li>采取在不同的递增的电压值的各个光谱，这取决于期望的分辨率这通常从百分之范围为一伏特的千分之几。通过使用自定义程序（ 例如，LabView的）相结合这些个别谱。 注:该方案可以使用范围广泛的不同的程序，以各个频谱的矢量列结合成一个矩阵很容易地进行编码，在所提出的实验这已被添加在缝合实时数据一起。 <ol><li>点击运行按钮采取偏见地图。这需要在光谱的一套偏见，使之成为列向量，然后将各加偏压谱的另一列。 注意:此生成数据马特里x，其中强度值对应于PL强度，行表示能量/波长，和列对应于电压。因为它是被运行，从而允许对数据的质量实时反馈偏置地图应该查看。 注意:偏压地图有助于识别不同的电荷的配置，并给出了正确的信息来完成激发和检测的路径两者的设置。 </li></ol></li></ol></li><li>标识会兴奋的过渡。注意转变的能量和所关注的偏压范围。 <ol><li>此时决定如何激光激发将通过过渡进行调谐。有三种不同的选择，以获得激光激发到基态跃迁: <ol><li>调由温度的跃迁能量。18 </li><li>使用过渡Stark位移与激光能量达到共鸣。19 注意:上面我所提到的两种方法的很好的功能在于不需要一个可调谐激光源，由于QDM跃迁通过固定激光能量调谐秒。 </li><li>另外，使用可调激光器光源，通过过渡步进激光能量。的检测信号将出现在激光谐振与过渡，这给测量其激光有限的光谱分辨率。这将是该协议的其余部分的焦点。 </li></ol></li></ol></li><li>与过渡确定和实验参数设定，选择用于测量两个激发和检测的能量。 <ol><li>选择激发能作为过渡。选择检测作为激发跃迁的能量减去纵向光（LO）与半导体合金相关联的声子的能量。使用这些值，选择测量的正常的边通滤波器;它们必须在激发和检测能量之间截断。 注:对于experimental结果提出，从动过渡是在图3所示的中性基态激子，在1,301.7兆电子伏观察和-1的LO声子发射位于1266兆电子伏，分别对应于952.5 nm和979.3纳米。因此，我们使用的激发一个960纳米的短通滤波器和用于检测960纳米长通滤波器。干扰截止滤波器是理想的用于此目的，因为它们可以通过调整角度来调节。 </li></ol></li><li>设置激光在感兴趣的跃迁能量，这是由激光控制软件的前面板在相应的输入字段中输入所需的激光波长值简单地进行激励。 </li><li>的中心波长设定为预定值并监控由分光计控制软件的前面板上的相应的输入字段中输入所希望的中心波长值的-1 LO光子发射。 注:的InAs / GaAs中-1 LO光子发射是APPRoximately 36兆电子伏的激子的过渡，这将是兴奋之下。 </li><li>使用相机软件，通过点击对焦模式按钮，运行在连续模式下的光谱采集软件采集开始与CCD。信号应该是可见的或可能仍然由激光散射被隐藏。 </li><li>最大化的信号。关键的一步:调整激发短通滤波器，通过稍微调节其角度，使其具有适当的波长截止。 注意:最佳角度是通过监视信号而调整所述短通滤波器的角确定。通过改变滤光器的角度这改变了截止波长。关键是要确保尽可能多的激光光尽可能从集合抑制。 </li></ol> 3.准谐振测量设置<ol><li>使用自定义软件的主屏电脑控制设定的实验参数。要做到这一点，开始收集程序，并单击次式步骤极化，温度，或WL标签。这将所有的实验值，一旦运行，通过各种参数收集数据。 注:对于我们的实验数据采取所有计算机对照组自定义编程。关键是要具有软件或程序能够设置光谱仪的中心波长，控制CCD和源表，并通过过渡步骤中，激光能量收集，同时范围在不同偏压频谱。 <ol><li>输入激光将扫描通过所建立的激光能量范围:从1,301.7兆电子伏的中性激子跃迁能量高于约50μeV，低于50μeV。设置初始波长开始使用"所需WL（nm）的"场扫描。设置激光的端部范围上扫描（"结束电机单元"）。 </li><li>设置源仪表将通过点击"电压设置"选项卡的扫描过偏置范围。设置开始偏向VALUE（"电压开始（V）"），则结束偏置值"电压完（V）"和偏压步长"电压工序（V）"。在这里，扫描的偏差范围为1.68〜1.82 V. </li><li>输入积分时间通过点击"相机设置"选项卡上选择。设置积分时间，在"曝光（S）"的CCD（参见步骤3.3）。选择合理的积分时间在CCD。实验用积分时间，获得良好的信号。积分时间越好信号平均即得到20越大。 注意:用于实验积分时间分别为10秒。但是，积分时间可以低至0.5秒，这取决于PL信号的强度。有时它甚至没有必要看到单独扫描，但在缝合光谱一起数据求和加眼睛内插将揭示在光致发光激发（PLE）地图的转换的能力的信号。 </li&gt; </ol></li></ol> 4.数据采集<ol><li>一旦实验参数建立开始实验。通过点击运行按钮开始采集。 注意:在每个激光能量的软件而异的偏压服用光谱和背景光谱。这是对每个偏压步骤完成。然后激光能量是变化的并且该过程继续进行，直到所选择的整个范围就完成了。 </li><li>后处理的数据。 <ol><li>采取以每隔偏压地图的端部所用的额外的背景扫描和从各偏压列减去平均值。使用背景减除计划，或者写一个程序，它在后台列，平均它们放在一起，减去该平均背景光谱从数据矩阵的每个偏差列。注意:请参阅补充编码文件在我们的实验室中使用的程序。 注意:这将删除造成的任何剩余散射的激光其他杂散信号，大大即时通讯证明了偏见地图。 </li></ol></li><li>分析数据。 <ol><li>例如，要提取的频谱线的特性参数使用一个数学拟合软件运行洛伦兹在PL图的每个偏压片嵌合。21一旦拟合程序已经完成所有的信息将在嵌合这样的系数作为最大强度，光谱位置和FWHM。 </li></ol></li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+dependent+photoluminescence+of+charged+quantum+dot+molecules.">Polarization dependent photoluminescence of charged quantum dot molecules.</a> Phys. Stat. Sol. (c). 5 (7), 2464-2468 (2008).</li><li>Jelezko, F., Wrachtrup, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+defect+centres+in+diamond:+A+review.">Single defect centres in diamond: A review.</a> Phys. Stat. Sol. (a). 203 (13), 3207-3225 (2006).</li><li>Doherty, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nitrogen-vacancy+colour+centre+in+diamond.">The nitrogen-vacancy colour centre in diamond.</a> Physics Reports. 528 (1), 1-45 (2013).</li></ol>

作者什么都没有透露。

The above instructions demonstrate the phonon-assisted quasi-resonance spectroscopy method. By exciting into a QD discrete state, one can monitor the phonon emission line, achieving high resolutions. In the example provided, by using phonons it is even possible to resolve the lifetime-limited linewidth of the neutral exciton visible in experiments. The method is easy to incorporate into existing PL spectroscopy setups. As mentioned, once the energy of the desired transition line is identified via non-resonant spectroscop...

高分辨率的关键是解锁新的知识。这方面的知识，新技术可开发诸如更好的传感器，更精确的制造工具，并且更有效的计算设备。产生该密钥，但是，往往是以牺牲资源，时间或两者成本很高。这个问题是从解决解除简并的电子自旋天文学用一个很小的光谱移动可导致探测行星遥远的恒星旁的原子物理学在所有尺度无所不在。1,2,3 这项工作的重点是使用标准的光谱仪的设置和显示它如何解决低于其分辨率极限的光谱特征，特别是关于半导体光学领域。给出的例子是在的InAs / GaAs量子点（QDs），该各向异性电子-空穴的（EH）交换分裂，这是几个μeV的量级。4 ​​分光计C的分辨率极限一个通过组合标准PL与激光光谱技术来克服。准谐振荧光的这种方法具有实现使用平常单级光谱激光分辨率有限的额外好处。 对于单一QD的PL光谱的标准光谱学系统由一个单级0.3-0.75米单色和耦合装置（CCD）检测器与激励激光源和光学器件沿收费。这种系统是在最好能在约950纳米的近红外光谱分辨50μeV的。即使使用的统计和反卷积技术，例如单个单色器的设置是不能够在PL测量解决小于20μeV5的这种分辨率也可以通过使用三分光计得到改善，在三重添加剂模式，其中，谱所有三个光栅陆续散去。三重光谱仪具有更高的分辨率的优势，能够解决的约10μeV。在可替换的配置中，三重减色模式中，第一两个光栅表现为一个带通滤波器，得到了能够由小于0.5兆电子伏的激发和检测分离的附加功能。三重分光计的缺点是，它是一个昂贵的系统。 呈现感兴趣的方法之前，我们简要讨论其他的实验方法，与增加的复杂度，实现更好的光谱分辨率，能够解决单个量子点的精细结构。这些方法的元素是相关的呈现方法。一种这样的方法是增加在单个分光计设置的检测路径的法布里-珀罗干涉仪（FPI）6使用该方法的分辨率由FPI的精细度设置。因此，该光谱仪的分辨率提高到1μeV，在增加了复杂性和较低的信号强度的成本。7所述的干涉仪的方法也可改变一般操作，实际在与CCD照相机分光计，有效成为单点检测器，并通过各种能量的调谐是通过调整FPI腔本身来实现的。 共振荧光（RF）谱，在一个单一的光学转换是既兴奋和监控还提供高分辨光谱的承诺的另一种方法。光谱分辨率仅由激光线宽的限制并保持CCD作为一个多通道检测器，其中，不只是一个传感器检测所述信号，但是许多的CCD像素。此多通道检测是在信号平均而言是有利的。在射频光谱的挑战是由散射激光的大背景在单一QD电平测量时分离PL信号，特别是。许多技术可用于降低信号的散激光，涉及任一极化8，空间9或时间分离10比的激发和检测。第一种方法是使用高消光偏振器以抑制散射光，但这种方法具有从PL失去偏振信息的不利结果​​。8另一种可能的方法来获得共振荧光工程师被耦合到光腔的半导体系统，其中所述激发和检测的路径在空间上是分开的。这消除了解决从大型激光背景下PL信号的问题。但是，该方法仅限于复杂的样品制备是在密集的一般资源9 另一个类的方法，这也是能够解决分钟能量差是纯激光光谱，诸如差分传输，其具有实现完全偏振信息的激光限定分辨率的好处的。这种方法通常需要锁定检测的反式观察微小变化任务信号相比，在大的激光的背景。11最近，先进的纳米加工导致的激光的，与QD（多个）到值高达20％交互的馏分升压，通过或者使用指数匹配的固体浸泡镜片或嵌入光子晶体波导的点。12 即使这些方法具有实现高能量分辨率的能力，他们来在昂贵的设备，复杂的样品制造和丢失信息的费用。在此工作的方法结合了这三种方法元素，而在仪器或样品制作到正规的PL设置增加复杂性。 最近的工作已经表明，在减色模式三重光谱仪系统，可以可视化的单线态-三线态精细结构的量子点的分子（QDM）的两个光子跃迁的频谱13的数量级上的参与能量分裂几到几十μeV的被使用的是三减色模式，该模式允许共振激发转换和内小于一个毫电子伏特检测解决。光谱信息是通过使用声子和其它躺在较低的激子跃迁监控过渡下方提取。也可以应用此方法来解决分别8μeV和4μeV，的激子跃迁的各向异性诶交换分裂，甚至在寿命有限的线宽， 如图1所示。这个结果类似，本文将集中于一个简单的光谱仪的设置，将包括许多与其他高分辨率的方法具备的优势。此外在CCD仍将作为一个多通道检测器。实验装置也可以相对于其他的高分辨率光谱学方法相当便宜保持和具有被容易地修改以实现单点相关测量的额外好处。不同于全光照的结果摹声子和三重光谱仪，底层的关键是要充分利用与半导体及相关合金构成的半导体样品相关联的LO声子卫星。的LO声子卫星和零声子线（ZPL）之间的能量分离是几十兆电子伏的这种样品的顺序上，允许使用一个单级光谱仪。14这种能量分离允许使用所提出的准通过共振驱动的过渡，并等于一个LO声子能量监测激发低于-resonance光谱法。这种技术是类似于PL激励的，其中一个激发到激发过渡并监视地面状态转换15的过渡之间的间隔被激发并且LO声子卫星的允许使用的边缘通滤波器来抑制弹性散射光。使用声子卫星的这种方法允许激光线宽限制的分辨率由于共振令人激动的过渡是典型的LO声子卫星发射变得可见唯一的一次。

<table><tbody><tr><td>Tunable Diode Laser DL pro</td><td>Toptica Photonics</td><td>DL Pro</td><td></td></tr><tr><td>Closed Cycle Cryogen Free Refrigerator System for Microscopy</td><td>Cryo Industries of America Inc.</td><td>Cryocool G2</td><td></td></tr><tr><td>Sourcemeter</td><td>Keithley&amp;nbsp;</td><td>2611a</td><td></td></tr><tr><td>50X Mitutoyo Plan Apo NIR Infinity-Corrected Objective</td><td>Mitutoyo America Corporation</td><td>378-825-5</td><td></td></tr><tr><td>Turbo pump</td><td>Pfeiffer Vacuum</td><td>HiPace 80</td><td></td></tr><tr><td>NIR coated Mirrors&amp;nbsp;</td><td>Thor labs</td><td>BB1-E03</td><td></td></tr><tr><td>Polarizers&amp;nbsp;</td><td>Thorlabs</td><td>LPNIR050-MP</td><td></td></tr><tr><td>200&amp;nbsp;mm AR coated Achromatic lens&amp;nbsp;</td><td>Thorlabs</td><td>AC254-200-B-ML</td><td></td></tr><tr><td>100&amp;nbsp;mm AR coated Achromatic lens&amp;nbsp;</td><td>Thorlabs</td><td>AC254-100-B-ML</td><td></td></tr><tr><td>960 Long pass filter</td><td>Thorlabs</td><td>960aelp</td><td></td></tr><tr><td>960 Short pass filter</td><td>Thorlabs</td><td>960aesp</td><td></td></tr><tr><td>Liquid Crystal Variable Retarder</td><td>Meadowlark Optics</td><td>LVR-100</td><td></td></tr><tr><td>0.75&amp;nbsp;m Spectrometer Acton SpectraPro</td><td>Princeton Instruments</td><td>Trivista</td><td></td></tr><tr><td>Liquid Nitrogen Cooled Camera&amp;nbsp;</td><td>Princeton Instruments</td><td>7508-0002</td><td></td></tr><tr><td>External Camera</td><td>Watec</td><td>Wat-902H Ultimate</td><td>Optional</td></tr><tr><td>Ostoalloy</td><td>Lake Shore Cryotronics</td><td>Ostalloy 158</td><td></td></tr><tr><td>Gold wire (40 gauge)</td><td>Surepure Chemetals</td><td>Au-Wire-03-02</td><td></td></tr><tr><td>Silver Epoxy</td><td>A.I. Technology</td><td>Prima-Solder EG8020</td><td></td></tr><tr><td>Program Software&amp;nbsp;</td><td>National Instruments&amp;nbsp;</td><td>LabView</td><td></td></tr></tbody></table>

high resolution phonon-assisted quasi-resonance fluorescence spectroscopy

高分辨率光学光谱方法都要求在任一技术，设备，复杂性，时间或它们的组合方面。在这里，我们证明的光学光谱方法，其能够使用标准的，易于使用的光谱仪的设置解决光谱特征之外，该自旋精细结构，单量子点（QDs）的均匀线宽的。该方法采用激光和光致发光光谱，结合多通道荧光检测激光线宽分辨率有限的优势。这样的方案允许在一个共同的单级光谱仪相当大的改进的分辨率。该方法使用声子协助在一个单一的量子点的光致发光的测定其基态跃迁的共振激发之后。声子的能量差允许一个分离并过滤出激光光激发量子点。一个有利的FE这种方法的ATURE是其直接的集成到标准的光谱设置，这对大多数研究人员访问。

在附图中呈现的结果表明，使用声子协助PL测定的高分辨率能力。的示意图（ 图2）表明，与在两个激发和检测的边缘通滤波器之外，实验装置仍然是一个标准光谱的设置，可任选地加入偏振控制的。跟单和三重分光计（ 图3）对比描绘了声子辅助方法的重大改进分辨率。各向异性诶分裂清楚地显示允许分裂（ 图4）的精确测量。该方?...

Watch this Scientific Journal Video about High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy at JoVE.com

High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

The manuscript describes a method of phonon-assisted quasi-resonant fluorescence spectroscopy that incorporates both laser-limited resolution and photoluminescence (PL) spectroscopy. This method utilizes optical phonons to provide linewidth-limited resolution spectra of atom-like semiconductor structures in the energy domain. The method is also easily realized with a single spectrometer optical spectroscopy setup.

高分辨率声子辅助准共振荧光光谱仪

High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we ...

high-resolution-phonon-assisted-quasi-resonance-fluorescence

Research

JoVE Journal

Engineering

7.4K 次观看.  University of California, Merced. The manuscript describes a method of phonon-assisted quasi-resonant fluorescence spectroscopy that incorporates both laser-limited resolution and photoluminescence (PL) spectroscopy. This method utilizes optical phonons to provide linewidth-limited resolution spectra of atom-like semiconductor structures in the energy domain. The method is also easily realized with a single spectrometer optical spectroscopy setup. 

视频: High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to &lt; 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

We discuss the construction and operation of a complex nonlinear optical
system that uses ultrafast all-optical switching to isolate Raman from fluorescence
signals. Using this system we are able to successfully separate Raman and fluorescence
signals utilizing pulse energies and average powers that remain biologically safe.

波谱与自发拉曼显微荧光背景的抑制

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ...

科研

生物工程

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

生物化学

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

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 &#181;g, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

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 ...

工程学

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or &#8220;tapping mode&#8221; AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.
PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.
The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

冲红灯光感受器原子力显微镜使用PeakForce定量纳米机械性能映射

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

单分子Fürster共振能量传递方法，用于GEN1对霍利迪结分辨率进行实时调查

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

遗传学

Resonance Fluorescence of an InGaAs Quantum Dot in a Planar Cavity Using Orthogonal Excitation and Detection

The ability to perform simultaneous resonant excitation and fluorescence detection is important for quantum optical measurements of quantum dots (QDs). Resonant excitation without fluorescence detection &#8211; for example, a differential transmission measurement &#8211; can determine some properties of the emitting system, but does not allow applications or measurements based on the emitted photons. For example, the measurement of photon correlations, observation of the Mollow triplet, and realization of single photon sources all require collection of the fluorescence. Incoherent excitation with fluorescence detection &#8211; for example, above band-gap excitation &#8211; can be used to create single photon sources, but the disturbance of the environment due to the excitation reduces the indistinguishability of the photons. Single photon sources based on QDs will have to be resonantly excited to have high photon indistinguishability, and simultaneous collection of the photons will be necessary to make use of them. We demonstrate a method to resonantly excite a single QD embedded in a planar cavity by coupling the excitation beam into this cavity from the cleaved face of the sample while collecting the fluorescence along the sample&#39;s surface normal direction. By carefully matching the excitation beam to the waveguide mode of the cavity, the excitation light can couple into the cavity and interact with the QD. The scattered photons can couple to the Fabry-Perot mode of the cavity and escape in the surface normal direction. This method allows complete freedom in the detection polarization, but the excitation polarization is restricted by the propagation direction of the excitation beam. The fluorescence from the wetting layer provides a guide to align the collection path with respect to the excitation beam. The orthogonality of the excitation and detection modes enables resonant excitation of a single QD with negligible laser scattering background.

Resonant excitation of a single self-assembled quantum dot can be achieved using an excitation mode orthogonal to the fluorescence collection mode. We demonstrate a method using the waveguide and Fabry-Perot modes of a planar microcavity surrounding the quantum dots. The method allows complete freedom in the detection polarization.

用正交激励和检测方法研究平面腔内铟量子点的共振荧光

The ability to perform simultaneous resonant excitation and fluorescence detection is important for quantum optical measurements of quantum dots (QDs). ...

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

超快时间解析近红外刺激拉曼测量功能 +偶联系统

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near ...

化学

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

高分辨率中子光谱研究蛋白质和水合水的皮秒纳秒动力学

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

生物学

Localized Surface Plasmon Resonance Imaging to Detect Protein Secretions from a Single Cell

Source: Raghu, D. et al., <a href="https://app.jove.com/v/53120">A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates localized surface plasmon resonance imaging to detect protein secretions from a single cell. Using a microfluidic setup and a peptide-bound plasmonic nanostructure chip, changes in reflected light intensity at the resonance angle reveal interactions with secreted proteins from a single cell.