我们非常感谢美国国家科学基金会，NSERC，美国国立卫生研究院和ONR。

背景： Apertureless探头近场红外显微镜提供高空间分辨率的成像。它是一个相对较新的技术，其中一个事件的红外光束被锋利的原子力显微镜（AFM）在接近样品的悬臂共振频率振荡提示分散。红外探测器收集的散射光，并在这个共振频率及其谐波解调。这样一来，其余样品表面上的聚焦激光束事件的背景分散可以降低一个远远超出了光的衍射极限的空间分辨率可以达到获得与纳米级空间分辨率的我 ，二，三红外对比。由于针尖的顶点是比较小的激光束的重点领域，散射光较弱。为了加强这方面的散射场，零差检测是用来参考字段被添加到收集的散射场的相对相位的领域是这样，最大的建设性干扰发生在探测器。散射光强度，然后参考电场IV，V，VI的大小成正比。在近场成像的一个重要的问题是，以避免针尖第七，第八，九，十 ，十一Z -议案生产的工件。这个问题可以减少零差阶段的适当调整，但不包括大的地形特点，穆勒等人证明，可靠地使用这种技术获得的空间分辨率小于 30纳米的15的实验材料散射光谱。近场显微镜的生物材料的使用已证明以前，特别是大分子，如烟草花叶病毒第十二和大肠杆菌第十三。 在这份报告中，我们说明了这样一个成像设备的组装。我们也出席了会议淀粉样纤维的形成apertureless扫描近场红外显微镜（ANSIM）＃21-31肽片段的β2 - M的二级结构信息。近场图像采集的同时，地形，使个别纤维的散射光谱探测和收集。 我们的apertureless近场扫描红外显微镜（ANSIM）测量的是一个自制的设备。实验装置的原理是在Scheme 1所示。原子力显微镜显微镜（多模，威科仪器，圣巴巴拉，CA）是用于测量样品的地形，以及生产的近场增强散射在尖端振荡频率调制。攻模式，NSC14/Ti-Pt铂金涂层的悬臂（MicroMasch爱沙尼亚），以提高散射连续的，可调谐红外激光（频率范围：2000厘米 -1到1600厘米-1，PL3二氧化碳气体激光，爱丁堡仪器，大不列颠）附近的表面。一个氦氖激光器（梅勒斯Griot，新墨西哥州）字段是用来作为一个不可见的红外辐射的指导。红外激光光源是传播传递ZnSe的部分反射后，对镜头。然后集中到振荡针尖的顶点，与探头长轴平行的光束的偏振。镜头收集红外辐射，然后添加一个参考零差信号。一个抛物面镜聚焦到一个汞镉碲（MCT）红外探测器（红外Graseby，佛罗里达州奥兰多市）的红外辐射。利用压电驱动器（Thorlabs，牛顿，新泽西州）是最大限度地纠正阶段的零差光检测到的信号。上文所述的光学元件大部分都是在一个平台上，紧紧贴在XY或Z位置使用平移阶段转移。交流的一小部分检测到的信号传送到锁相放大器（SR844模型，斯坦福研究系统，加利福尼亚州桑尼维尔，射频）信号解调提示振荡频率。散射强度观察，针尖扫描采样表面的地形数据是同时获得。数据和图像采集所用的软件是奈米级V5.31r1（Veeco Instruments公司，圣巴巴拉，CA）。 计划 <img src="/files/ftp_upload/1581/scheme1.jpg" alt="方案1" /> 图1 -显示成像系统部件的组装和使用，我们将描述 <img src="http://jove.com/files/ftp_upload/1581/1581fig0.jpg" alt="图1" /> 0）光学表 1）基地的MM原子力显微镜 2）MM的原子力显微镜的扫描仪 3）高架光学面包板 （13“× 18”× 3 / 8“，Thorlabs） 4）指导镜子（镀金，1“直径） <stro吴&gt; 5）光管与虹膜和中心块 6）光学的多维数据集W / ZnSe的部分反射 7）光电管，用于确保升降平台上的安装（ 17） 8）红外目标 （NA 0.28，伊林佛罗里达州16毫米，） 9）收集回来的散射光的光管 10）立方W /离轴抛物面镜 （90 °，佛罗里达州5“，亚诺什） 11）立方举行GE板@ 45 ° 12）显微镜目镜（X10） 13）XY阶段安装针孔（0.5毫米） 14）的MCT红外探测器（Graseby红外） 15）红外探测器前置放大器（AC和DC） 16）XY工作台移动光束位置 17）升降表，以集中光束 18），Z级定位红外探测器 19）的X期位置的红外探测器<img src="/files/ftp_upload/1581/1581fig1.jpg" alt="图1" /><center>一，最初的装配的光学装置</center>大会一步一.1 装配的光学装置和调节后视镜和位置的氦氖（氦氖）束平行光表约正确的高度以上的光学实验电路板（3）和距离（D）（H）从中心（0）扫描仪（2）。 这个高度H和距离 D可估计从样品的高度，从光学扫描器表（2），所需的入射角（α），实验电路板的顶部的高度以上的光学表上（H1） （H），光立方体和红外目标（图1 6和8）和红外目标的工作距离（ 所有D，这里D = 1“2”2“）的几何尺寸 图2 <img src="/files/ftp_upload/1581/1581fig2.jpg" alt="图2" /> H = H1 + D *罪（α）- H D = D * COS（α） 装配步骤.2 图3 <img src="/files/ftp_upload/1581/1581fig3.jpg" alt="图3" /> 图3：在立方体分光镜（5）没有拉长光管在多维数据集（6）的另一端直接通过氦氖束管的两端连接的虹膜。 大会步骤.3 图4 <img src="/files/ftp_upload/1581/1581fig4.jpg" alt="图4" /> 图4：与扫描仪（2）删除，重视与红外目标的虹膜长的光管。插入部分的硒化锌反射沿着长的光管，直接走向样品的光束。 ZnSe的部分反射应安装这样的氦氖光束击中的前表面反射光学控股的部分反射立方体的几何中心。 图5 <img src="/files/ftp_upload/1581/1581fig5.jpg" alt="图5" /> 图5：通过旋转的部分反射，直接通过封闭的输出虹膜的氦氖光束。由于部分反射镜的安装并不持有完全垂直，安装有两个调整螺丝，允许梁的垂直运动。如果有必要使用这些。 图6 <img src="/files/ftp_upload/1581/1581fig6.jpg" alt="图6" />大会步骤.4 图6。安装抛物面镜光立方体的橡胶O型圈（参见图1，图5）。通过拧紧螺丝，O型圈压缩，使后视镜调节。 图7 <img src="/files/ftp_upload/1581/1581fig7.jpg" alt="图7" />附加镜（一种是如图7所示）直接在相反方向的氦氖的光束，如图7所示。调整光束通过以前使用的虹膜。这束将用于调整抛物面镜。 图8 <img src="/files/ftp_upload/1581/1581fig8.jpg" alt="图8" /> 图8：一个抛物面反射镜放置在光学的多维数据集（10）反映朝着探测器的位置。调整螺丝直接通过针孔放置在多维数据集（11），将持有的锗（Ge）窗口的输出光束。后束是通过针孔调整，放置在MCT ðetector关闭针孔。调整的红外探测器等，氦氖光束探测器的传感元件是的位置。移动探测器下降1〜2毫米（红外线光束将被转移由GE窗口）。 大会一步一.5 图9 <img src="/files/ftp_upload/1581/1581fig9.jpg" alt="图9" /> 图9：与GE窗口插入到光学的多维数据集（11）坐骑。涂上防反射（AR）的葛窗口作为红外过滤器，并允许目视观测的悬臂和样品。将目镜（12）。扭到多维数据集的多维数据集（11）角度（10）允许一个指向所需方向的目镜。氦氖光束通过旋转葛窗口装载，直接通过目镜中。 图10 <img src="/files/ftp_upload/1581/1581fig10.jpg" alt="图10" /> 图10：连接红外目标（8）和原子力显微镜扫描仪（2）。附加的beamstop光管（5）的输出端。氦氖光束通过目镜观看，使用保护性过滤器。样品扫描仪上的位置，并直接通过输入光管（5）氦氖光束。确保氦氖光束足够宽，溢出的beamstop。 beamstop是因为卡塞格林目标，其中仅收集入射光束的外围。通过调整目镜的线程，一个侧重于样品的氦氖束锐利的图像。 大会一步一.6 一个抛物面镜的最后的调整是必需的。输出针孔的抛物面镜的焦点位置的虹膜代替。删除葛窗口和红外探测器（红外探测器的位置标志！）。镜头焦距的大约从虹膜虹膜后附加一个额外的镜头。在这一刻，从事悬臂应通过镜头可见。调整的抛物面镜中心通过封闭的虹膜提示的位置。需要注意的是正确的氦氖光束集中到尖端时，它使明亮旌宇之间的针尖与样品的表面上它的反射。重新连接GE窗口;调整的悬臂，方便直观地观察它。更换针孔的光圈，并记住，用于红外探测针孔应移出中心由GE窗口的红外线光束位移。红外探测器放置在前面的显着位置。 最后，直接红外线光束，使同行可见的氦氖用于调谐梁。 现在，一切都应该是准备进行例行调整。 常规调整： 调整步骤A.1： 与氦氖激光对准 它比可见的氦氖（632nm）与不可见的红外线（约6μm）配合。 氦氖和红外光束的路径走到了一起，在可倾斜的镜子。如果这面镜子是倾斜的氦氖可以通过，如果镜子中的地位是的红外线光束传播到近场的设置。仅用于校准梁之一，使用两镜在共同与其他的路径，均位于前可倾斜的镜子。如果您不小心移到另一个镜像，第一次尝试，使这面镜子，在旧的位置。 第A.1.1条。粗对准两镜使用氦氖激光的两镜对准光束通过近场阶段沿路径的虹膜。如果你看一下在零差臂梁，电晕般的光束轮廓，应注意观察（由于束在光管受体阻滞剂）。这表明，电子束直接通过管道传递。 A1.2。近场阶段的精细对齐将原子力显微镜头，并参与到样品的悬臂尖端。氦氖光束聚焦到成像悬臂结束。如果这样取代从中间的输入光管（5）重复调整步骤A.1梁。 A.1.2。直到梁为中心。 通过移动平移光学阶段，反射显微镜的目标是调整，把重点放在悬臂尖端的顶点，它是分散的光。 旋转低于目镜葛镜（位于光立方体），并期待通过目镜。束聚焦在这个点由抛物面反射镜（或红外探测器的立方体时，葛镜拉）。向前或向后移动舞台，针尖与样品表面的反射，直至观察到清晰的图像。使用中的两个o有方向（上/下和左/右）的地方，大约在目镜中的针尖。 透过望远镜，观察原子力显微镜的悬臂，其尖端。同样，请记住使用保护的过滤器，当观看氦氖光束。没有氦氖光束，一些红灯还是从内部的原子力显微镜远程控制光的起源。在右左和/或上下方向移动，直到鲜红的旌宇观察针尖上的平移阶段。如果校准是相当糟糕，移动舞台的原子力显微镜的左侧，慢慢向右移动。观察反射样品表面上的一个红色的移动反射。如果仍然没有观察到红色的反映，是舞台，翻译左再移动上下的时尚。保持左，右，上下翻译，直到一个红色的旌宇观察氦氖光束集中在针尖的顶点。 A1.3。重叠零差场散射光打开零差臂，并期待彻底目镜。降低强度在三个或更多点看到，这些斑点是前端和背部不同的光学多次反射的结果。在零差臂移动的镜子，让第二的位置（从底部和强度）的一角，这是提示和它的反射走到一起的美好形象重叠。 A1.4。位置探测器删除葛镜和氦氖光束红外探测器是位于方向走。背后的光管，两个环形斑。第二点（强度）是现货，应该去面对红外探测器的热保护箔孔通过。当场强度最高的应该被看作孔的边界。 A1.5。更改从一个样品到另一个更改后的样品针尖不再像以前一样的确切位置，但不应该太离谱。步骤A.1.2启动以来所不同的是通常不是很大。 注意：如果校准是关闭的，请检查如果氦氖光束仍能通过，所有的虹膜和针尖上创建了一个明亮的红色旌宇。有时候，你不通知你有感动，因此移动从其原始位置的一面镜子。如果对齐方式仍然是坏的，不幸的是，整个校准程序再次完成。 调整第2步：对准红外线光束使用CO激光线与高强度/功率（至少100毫瓦），因为这会更容易对齐。用液态氮填充探测器，让它平衡至少30分钟。 2.1。粗对准两镜广场后面的第一个虹膜的功率计监测传入的红外线光束的功率。其次，调整，这是位于前可倾斜的镜子的镜子，以获得最高的的功率读数。功率计和虹膜后面的这是最接近近场阶段，调整的可倾斜的镜子，直到获得最大功率读数举行。重复此步骤，优化调整了几次。 2.2。看着1F信号参考锁相放大器必须设置隔离1F（分机），这是原子力显微镜的振荡频率设置提示振荡频率的频率。设置生效至3微米的奈米级软件的情节模式的扫描尺寸。 2.1粗调后，寻找1F信号的形状。其次，通过调整压电驱动器的提示和homodyned光从零差臂从收集到的散射光之间的调整阶段。这驱动器在零差臂通过改变压电电压，使1F信号最低为500纳米左右，从零起步的一面镜子。因为在走线的每一次变化会改变两个光路，因此它们的相对阶段的长度，相需要与压电纠正。 <table border="0"><tbody><tr><td colspan="2" style="font-weight:bold;">你会看到（1F信号）？ </td></tr><tr><td> 坏对齐： </td><td>将观察到3微米的Z扫描大小颠簸。 </td></tr><tr><td> 中等对齐： </td><td>第一凹凸曲率看起来有点超过凸凹，和第二个凸点是比第一次小。 </td></tr><tr><td> （几乎）都配合得很好： </td><td>将观察到两个颠簸。第一凸点高于第二个凸点，第一凸点右侧的曲率将是负面的（凹）。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2" style="font-weight:bold;">对齐怎么办？ </td></tr><tr><td> 错误的对齐方式： </td><td>根据需要重复步骤2.1校准程序。由于红外光束直径大，梁的大部分将通过虹膜，即使是不完全一致。即使两个颠簸仍在观察，1F信号量可以增加调整一个或两个对齐镜子。观察第一凹凸曲率的轻微变化。最重要的是，要有耐心，因为这是最困难的部分对齐。 </td></tr><tr><td> 中等对齐： </td><td>尝试调整一个或两个对准镜子获得了近良好的对齐。尝试以及移动XYZ平移阶段，但非常小的增量调整。 </td></tr><tr><td> （几乎）都配合得很好： </td><td>调整镜子，并设法增加第一凸点的最大。一楼的信号通常是8至16 V左右，低输入功率则较少。更改通过改变电压，使用第一个最小的压电驱动程序，以便更接近零的阶段。如果有一个重大1F信号，开关锁定放大器的参考隔离2F信号。信号的一些应注意观察，并尝试改善非常轻微调整的镜子和不断变化的阶段多一点点。 </td></tr></tbody></table> 2.3。更改从一个样品到另一个执行步骤下的氦氖光束对准A.1.5。搞针尖，并通过显微镜传播的红外线光束。观看1F信号。如果1F信号仍然是观察，调整的可倾斜的镜子和调整阶段。一个良好的中频信号，因此一个良好2F信号可能仍有待观察。如果没有，精细对准的光束，使用步骤2.2。 代表性的成果： 在匹兹堡大学和纯化（&gt; 95％）的高效液相色谱大学生物技术和生物工程研究中心＃21-31肽合成样品制备。 第十四合成淀粉样纤维，0.8毫克（Sigma - Aldrich公司）TMAO添加到1毫米的解决方案＃21-31肽在18MΩ的水，由杨等人执行的程序类似。 Ultraflat黄金基板第十五发了言。为期一个月的老解决方案（室温孵育，pH值5.5）40μL沉积到新鲜ultraflat黄金基板的几分钟。他们简要地流的18MΩ的水冲洗， 流动的N 2气体干燥，在ANSIM仪器定位。 图11显示为＃21-31肽纤维收集到的地形和近场图像。一）地形图像的同时获得其相应的近场图像。标签代表个人纤维和使用功能的二级构象类型，每个须根。 B和C）通讯近场图像收集在两个不同的波数：1631和1691年厘米-1。每幅图像的面积是1 × 1 微米 2 。左边刻度代表的高度，正确的规模是从分散的领域的锁相放大器。 图11 <img src="/files/ftp_upload/1581/1581fig11.jpg" alt="图11" />

<ol>
	<li>Mueller, K., Yang, X., Paulite, M., Fakhraai, Z., Gunari, N., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+imaging+of+the+surface+of+self-assembled+polystyrene-b-poly(methyl+methacrylate)+diblock+copolymer+films+using+apertureless+near-field+IR+microscopy.">Chemical imaging of the surface of self-assembled polystyrene-b-poly(methyl methacrylate) diblock copolymer films using apertureless near-field IR microscopy.</a> Langmuir. 24, 6946-6951 (2008).</li><li>Lahrech, A., Bachelot, R., Gleyzes, P., Boccara, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared-reflection-mode+near-field+microscopy+using+an+apertureless+probe+with+a+resolution+of+lambda/600.">Infrared-reflection-mode near-field microscopy using an apertureless probe with a resolution of lambda/600.</a> Opt. Lett. 21, 1315-1317 (1996).</li><li>Taubner, T., Hillenbrand, R., Keilmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+visible+and+mid-infrared+scattering-type+near-field+optical+microscopes.">Performance of visible and mid-infrared scattering-type near-field optical microscopes.</a> J. Microsc. 210, 311-314 (2003).</li><li>Kim, Z. H., Leone, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization-selective+mapping+of+near-field+intensity+and+phase+around+gold+nanoparticles+using+apertureless+near-field+microscopy.">Polarization-selective mapping of near-field intensity and phase around gold nanoparticles using apertureless near-field microscopy.</a> Optics Express. 16, 1733-1741 (2008).</li><li>Bridger, P. M., McGill, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+nanometer-scale+optical+property+discrimination+by+use+of+a+near-field+scanning+apertureless+microscope.">Observation of nanometer-scale optical property discrimination by use of a near-field scanning apertureless microscope.</a> Opt. Lett. 24, 1005-1007 (1999).</li><li>Stebounova, L., Akhremitchev, B. B., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+weak+scattered+signal+in+apertureless+near-field+scanning+infrared+microscopy.">Enhancement of the weak scattered signal in apertureless near-field scanning infrared microscopy.</a> Rev. Sci. Instrum. 74, 3670-3674 (2003).</li><li>Akhremitchev, B. B., Pollack, S., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apertureless+Scanning+Near-Field+Infrared+Microscopy+of+a+Rough+Polymeric+Surface.">Apertureless Scanning Near-Field Infrared Microscopy of a Rough Polymeric Surface.</a> Langmuir. 17, 2774-2781 (2001).</li><li>Hecht, B., Bielefeldt, H., Inouye, Y., Pohl, D. W., Novotny, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facts+and+Artifacts+in+Scanning+Near-Field+Optical+Microscopy.">Facts and Artifacts in Scanning Near-Field Optical Microscopy.</a> J. Appl. Phys. 81, 2492-2498 (1997).</li><li>Labardi, M., Patane, S., Allegrini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artifact-free+near-field+optical+imaging+by+apertureless+microscopy.">Artifact-free near-field optical imaging by apertureless microscopy.</a> Appl. Phys. Lett. 77, 621-623 (2000).</li><li>Palanker, D. V., Simanovskii, D. M., Huie, P., Smith, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+Contrast+Parameters+and+Topographic+Artifacts+in+Near-Field+Infrared+Microscopy.">On Contrast Parameters and Topographic Artifacts in Near-Field Infrared Microscopy.</a> J. Appl. Phys. 88, 6808-6814 (2000).</li><li>Akhremitchev, B. B., Sun, Y., Stebounova, L., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Monolayer-Sensitive Infrared Imaging of DNA Stripes Using Apertureless Near-Field Microscopy.Langmuir. 18, 5325-5328 (2002).</li><li>Brehm, M., Taubner, T., Hillenbrand, R., Keilmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+Spectroscopic+Mapping+of+Single+Nanoparticles+and+Viruses+at+Nanoscale+Resolution.">Infrared Spectroscopic Mapping of Single Nanoparticles and Viruses at Nanoscale Resolution.</a> Nano Lett. 7, 1307-1310 (2006).</li><li>Dazzi, A., Prazeres, R., Glotin, F., Ortega, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+nano-chemical+mapping+performed+by+an+AFM-based+("AFMIR")+acousto-optic+technique.">Analysis of nano-chemical mapping performed by an AFM-based ("AFMIR") acousto-optic technique.</a> Ultramicroscopy. 107, 1194-1200 (2007).</li><li>Yang, D. S., Yip, C. M., Huang, T. H. J., Chakrabartty, A., Fraser, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+the+Amyloid-&#x03B2;+Aggregation+Pathway+with+Chemical+Chaperones.">Manipulating the Amyloid-&#x03B2; Aggregation Pathway with Chemical Chaperones.</a> J. Biol. Chem. 274, 32970-32974 (1999).</li><li>Meadows, P. Y., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+Microscopy+Studies+of+Fibronectin+Adsorption+and+Subsequent+Cellular+Adhesion+to+Substrates+with+Well-Defined+Surface+Chemistries.">Force Microscopy Studies of Fibronectin Adsorption and Subsequent Cellular Adhesion to Substrates with Well-Defined Surface Chemistries.</a> Langmuir. 21, 4096-4107 (2005).</li></ol>

assembly, tuning and use of an apertureless near field infrared microscope for protein imaging

本文旨在指导读者在超越衍射极限的成像的红外线近场显微镜的装配和操作。 apertureless近场显微镜是一种光散射式仪器，可提供大约20 nm分辨率的红外光谱。一个组件的完整列表和使用一步一步的协议提供。大会和仪器调谐常见的错误进行了讨论。一个有代表性的数据集，显示了二级结构的淀粉样原纤维。

Watch this Scientific Journal Video about Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging at JoVE.com

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

描述成像蛋白质总量的大会，是一个近场红外显微镜。

大会，调整和使用蛋白成像Apertureless近场红外显微镜

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The ...

assembly-tuning-use-an-apertureless-near-field-infrared-microscope

Research

JoVE Journal

Biology

9.5K 次观看.  University of Toronto. 描述成像蛋白质总量的大会，是一个近场红外显微镜。

视频: Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

代骨髓衍生的双光子成像使用的小鼠树突状细胞

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

科研

生物学

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

光镜下的主要组成部分

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

近红外光谱技术在婴儿，儿童及成人的认知功能探索

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

自动疏水层析蛋白质纯化用柱选择

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.