Name: high-resolution thermal micro-imaging using europium chelate luminescent coatings
Uploaded: 2017-04-16T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings at JoVE.com

Work at Argonne National Laboratory was funded by the Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357, which also funds Argonne's Center for Nanoscale Materials (CNM) where the patterning of the BSCCO mesa was performed. We thank R. Divan and L. Ocola for their help with sample fabrication.

1.样品用于涂料的制备注:如果可能，从样品的表面去除所有有机物污染以热成像。任何这样的污染可能与沉积的EuTFC膜反应并改变其发光响应，在所得到的热图像引起依赖于位置的伪像。这是与用Au的表面电极，其倾向于吸引从大气中有机污染物的样品特别重要的。除去同时坐在样品表面上的任何颗粒或灰尘，因为这些可能会导致伪像也。笔者建议采用以下步骤: <ol><li>使诸如超导桥或电阻装置的电流和电压连接，以在样品上的装置（ 例如引线键合，涂上使用导电环氧树脂等连接）清洗它在用于薄膜涂层制备之前 ，因为这些步骤可能引入污染w ^HICH应涂层前被去除。如果可能，请使用Au线，因为这将使它更容易在样品连接到低温恒温器的膜已经沉积之后。 （参见下面的步骤4.6）。 </li><li>清洗，在100％丙酮中的样品中的15秒的超声浴。 </li><li>而不使样品干燥，清洁它在100％异丙醇中进行5秒的超声浴中。 </li><li>使用氮气枪吹样品干燥。 </li><li>如果可能的话，则清洁使用氧等离子体灰化掉所述样品表面上的任何残留的有机残基。要做到这一点，使用100 W，22 立方厘米 /秒的O 2流量和160毫乇的气体压力的等离子体功率，60秒。为了避免样品的再污染，在该步骤之后尽快沉积EuTFC涂层。 </li></ol> 2.制备涂料体系的EuTFC沉积<ol><li>使用由从不锈钢储物柜制成的专门建造的船20×10×10mm的3大小时（LXWXH）的升华的源钢铁箔包封锰铜的电阻丝的线圈10，用于操作在约100 - 200℃。通过浸泡在丙酮中，因为这些将不利地新的膜的性能影响溶解从船上EuTFC的任何熔化的残余物。 </li><li>冲洗异丙醇船。 </li><li>允许船在进行EuTFC加载到它之前在空气中完全干燥。 </li><li>从水蒸汽和轻保护EuTFC粉末被存储，同时它。用玛瑙研钵，以消除任何可见肿块彻底粉碎EuTFC粉末。 注意:即使当粉末从水蒸气的保护，但它仍然可以结晶成100微米直径或更大的块状物。这些必须去除，因为它们将导致严重的非均匀膜时升华，导致在热图像伪影。 </li><li>安装样品支架和升华源在所述真空涂层系统，使得样品位于大约10mm源舟正上方（合适地取向的晶体厚度传感器，用于监控沉积速率）。连接源舟加热器导致其相关的真空馈通。 </li><li>填充源舟约2/3满大约0.2克地面EuTFC粉末。 </li><li>安装样品倒置直接在源舟以上（以确保沉积的膜的均匀性），优选使用双面胶带或粘点，而不是真空润滑脂，它可能污染膜。 </li><li>以减少样品表面和EuTFC粉末在大气中（特别是水蒸气）的曝光尽快使用旋转泵开始将淀积室抽真空。 </li></ol> 3.沉积EuTFC薄膜由热升华<ol><li>沉积室泵送至3×10 -5毫巴或更小，优选地使用涡轮分子泵。 </li><li>程序的体厚度监控仪来读取1.50克/厘米3的膜密度。 </li><li>应用0.5 W的功率到源舟加热器，轻轻地暖源，直到EuTFC开始升华。这将需要2 - 3分钟的厚度监测开始阅读一个明显的沉积速率。 </li><li>调整加热器功率来维持6的沉积速率 - 7纳米/分钟。只作小，速度慢的调整，因为沉积速率通常需要1 - 2分钟，在输入功率的变化做出反应。 注:小船温度足以在该结构中以沉积大于10nm /分钟可能导致粉末在船熔化，大大减少其表面积，因此升华速率。更重要的是，过多的舟的温度可能从化学上改变EuTFC并且因此强烈地降低其发光的热灵敏度。 </li><li>膜沉积的200纳米（通过厚度监视器读取）之后，关闭电源源。 （±20nm的是可以接受的在这里，虽然厚度在该范围之外显著将导致较低的胶片感光度）。 </li><li>厚度监视器上的读数达到零之后，排气腔室中，用干燥的氮气。取出后，在真空干燥器中不透光容器尽快避光和水蒸汽的样品，通过存储。 注意:这将分别防止EuTFC薄膜的漂白和化学降解。 </li></ol> 4.安装在测量低温恒温器样品<ol><li>放置真空润滑脂的斑点在中心低温恒温器样品台大约1-2毫米的直径。使用的样品台，其包括具有圆形顶面15毫米的直径铜冷手指。 注意:这是足够的尺寸以确保阶段和当样品被向下压平在其顶部上的样品之间的强热接触。 </li><li>如果样品基体是导电，通过放置一个10微米的Mylar片上的润滑脂的顶部从舞台隔离它，和一个第二大小同样在聚酯薄膜的顶部ð团块。 注:作者发现，最好是采用脂具有相对高的粘度（ 例如，有机硅系高真空油脂）比专门散热化合物，后者通常含有可在流到顶表面低粘度成分样品并污染它的EuTFC涂层。 </li><li>按样品向下使用镊子以同时施加力，两个对角相对的角的润滑脂的顶部，然后在适当位置夹住至少两个角部，使用黄铜螺钉和铍铜夹具。 注:如果在样品未牢固保持在适当位置，那么当电力被施加到它，使所得到的图像难于分析它可能漂移显著相对于所述显微镜。 </li><li>进行任何必要的电连接，例如用于电流和从样品到低温恒温器布线电压引线，注意不要让污染（ 例如焊剂的液滴）降落在EuTFC电影。 注:通过使用将做的工作，最好避免使用助焊剂这一步，在所有通量只有最小量做到这一点。如果Au线被用于连接到样品通量不应该是必要的。 </li><li>安装在它的xyz平移台上的样品的低温恒温器的显微镜下，安装其热屏蔽和光学窗口，并用涡轮分子泵抽空其样品的空间。 </li><li>覆盖低温恒温器的光学窗口用一块铝箔（或类似）的，以防止EuTFC的漂白通过在室内环境照明。小心不要损坏或执行此操作时污染显微镜镜头。 </li><li>冷却低温恒温器感兴趣的浴温度。对于本文描述的样品中，这通常是5 K和100之间K. 注意:不要让样品台坐125 K和175 K的温度长时间，因为在此范围内EuTFC影片最终会在结晶与不均​​匀发光特性也可随时间漂移一个多粒状的状态。通过该温度范围内冷却以2 K /分钟或更快的将确保不会出现此问题。如果低温恒温器在该温度范围意外地留时间太长，则EuTFC膜可以是可重复地通过简单地加热低温恒温器，以至少190 K中5分钟的复位"。 </li></ol> 5.收集热图像数据的<ol><li>安装在照明光学部件路径500纳米的截止波长的短通滤波器。 </li><li>安装用通带的中心波长= 610纳米，且FWHM = 10nm的带通滤波器，在收集光学器件的路径。 注意:通带窄是有利这里，因为它最小化的这有助于噪声，但不与信号背景光集合。该过滤器还必须选择以最小化它们之间的光谱串扰。 </li><li>允许光源升温，在我稳定TS稳态工作温度，并让相机冷却到它的平衡的工作温度。这大概需要30分钟两种情况。 </li><li>与所有的滤光器代替（由于聚焦位置是依赖于波长的）照射样本并对齐和显微镜聚焦到感兴趣区域。 注意:当不被成像的样品，使用或类似的快门，以避免EuTFC膜的样品和所得漂白的不必要的照明。 </li><li>收集的参考图像与施加到样品零电流。当收集的每个图像中，为暗计数，这可以从像素强烈变化到像素，以及给予一个显著偏移来自发光信号中的正确图像计数的校正。 注意:使用将取决于实验的要求的曝光条件（见讨论），但选择曝光条件，使得图像不包含饱和像素是很重要的。该需要参考图像由于所收集的发光强度通常会有所不同强烈依赖于样品的表面反射率，即使在其温度是完全均匀的。 </li><li>应用电偏压施加到样品中，相同的曝光条件作为基准下收集的图像，并且计算这些强度比。注:电偏压所需要的电平，其上正在研究的装置和自热行为的组合强烈依赖。这里提出的实施例典型地从几十mA，导致跨器件的偏压的几伏的数量级的样本偏置电流导致。 注:如果在样品显著相对于参考图像移动，则该像素数据应当被移动以进行补偿。 （然而，取决于相机的性能，这种转变可能在其对光的敏感度，这就是为什么该样品的运动应该如果在所有POS被最小化引入从像素到像素变化的噪声锡布尔赫丁）。如果需要在温度测量高的绝对精度，在灯强度的小的漂移可以被用于通过归一化图像到参考比校正为1中的样品（ 即 ，一个适合的区域，该区域足够远从自加热装置为能够不受其影响）。 </li><li>重复步骤5.6对所有感兴趣的偏置条件，同时保持浴温度恒定。 </li><li>所有感兴趣的浴温度重复步骤5.4至5.7。 注:根据低温恒温器，样品可能需要重新调整，并在每一个新浴温度重新聚焦。 </li></ol> 6.校准结果的<ol><li>收集零施加电流足以覆盖所关注的整个温度范围内的参考图像。 3〜4的图像在各温度下将足以建立的再现性，而为20K间距会给足够的数据点，以产生一个准确的校准曲线。 （参见图1b &lt;/强&gt;）。 </li><li>从该曲线，转换归一化强度图像转换成温度图。虽然绝对发光强度样品的局部表面反射率在很大程度上取决于其相对于温度的标准化行为只有通过这种非常弱的影响。 </li></ol> 7.样品储存和电影再利用<ol><li>与往常一样，继续由环境光保护漂白电影。注意:如果需要，在样品的EuTFC涂层能够承受重复的热循环，并保持在高真空时，其特性将保持稳定历时2-3周。 注:然而，储存在室温下高真空，即使，影片将降低超过2-3个月。 （变色和该膜的粗糙化可以在光学显微镜下容易看到）。如果这发生在这需要额外的热图像的样品，然后清洁该膜并更换它按照步骤1〜3。 </li></ol>

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The authors have nothing to disclose.

这表现在我们的研究结果，在本文中描述的技术产生的微器件的高分辨率热图像，具有良好的灵敏度，并且仅使用简单的光学显微镜设备。这相对于替代方法的技术（下面将要讨论的）的优点是最强的在大约250 K和下面，这意味着它的最重要的应用可用于研究，其被设计成在低温浴温度下操作的设备的自加热。这些包括超导电流磁带（其中骤冷成核是关键工程感兴趣的），用于光学检测窄带隙半?...

许多电子装置经历强烈的自我加热时电偏置到它们的正常操作条件。这通常是由于低导热性（例如，在半导体）和高功率消耗密度的组合。此外，在具有半导电状电阻器件（ 即具有∂ρ/∂Ť&lt;0）人们早已知道，存在局部的热失控的在一定的偏置条件1,2的可能性，其中，所述偏置电流流过不是均匀地通过该装置，而是在其与高度局部化的自加热有关，通常在微米的尺度窄的细丝。 了解这种自热物理学可能在某些情况下是必不可少的用于优化特定装置的设计，这意味着用于成像技术的温度上微米尺度是很有用。已经有最近的从技术发展的两个领域中这种技术的兴趣死灰复燃。其中第一项是用于在高温超导带成像淬火工艺，其中热显微成像允许淬火成核位点进行识别和研究3,4。所述第二应用程序是用于在叠层固有的约瑟夫逊结太赫兹光源，这是从的Bi 2的Sr 2 CaCu 2 O 8制成理解自加热。这些具有低的热导率和沿着电流流动的方向相关的半导体状的导电性（ 即，它们的晶体C轴）的组合，如上所述。它们不仅显示出实验复杂不均匀的自加热行为5，6，7，8 </sup&gt;，9，10，11就已经理论上预言，这可能是太赫兹功率发射12，13是有益的。 许多技术存在用于在微观长度尺度的样品的温度成像。最初采用这里所描述的热致技术用于半导体器件接近室温14，15，16但最近在低温浴温度下大于3，4，10，11中所述的超导条带和太赫兹源得到了应用。在CCD相机的分辨率和信号与噪声的性能提升已经使相当大的性能改进这一技术在过去的几十年。欧盟配位络合物铕thenoyltrifluoroacetonate（EuTFC）具有旋光发光是强烈依赖于温度的。在这个复杂的有机配体有效地吸收在宽带围绕345nm处的UV光。该能量通过分子内激发转移辐射较少的铕离子，其通过发光光子的在612纳米发射的复杂返回到其基态。强温度依赖性源于能量转移过程 17为使涂有该材料的物体的敏感的热探针。当涂层是激发近紫外源 - 诸如汞短弧灯 - 具有较低发光强度的区域对应于更高的局部温度。将得到的图像通过光学显微镜的分辨率和LUM的波长在空间分辨率限制inescence（在实践中，围绕1微米）。取决于所要求的信噪比，时间分辨率仅由照相机的快门速度由发光（不超过500微秒）15的衰减时间限制，并且更根本。这些特性使该技术器件温度的非常快的探测器，其产生直接的温度测量，使用相对简单和经济的设备。 这种技术被其它基团发表在过去的变化已采用小的浓度溶解在聚合物膜和旋涂到该样品表面3,4铕螯合物。这导致了涂层，该涂层是高度均匀的局部，但其具有在样品形貌的步骤显著厚度变化 - 如通常发生在微型 - 导致在发光响应WH强的空间变化ICH可以给图像伪影。我们在此描述的技术的变化采用热升华在真空中。这不仅避免了宏观膜厚度变化的问题，但每单位面积实现的更高浓度EuTFC提高显著的灵敏度并且降低了图像采集时间。一个相关技术采用SiC的涂层颗粒的表面，而不是EuTFC 7，8，9上。碳化硅提供温度灵敏度可比这里描述的EuTFC涂料，但该颗粒的尺寸限制了产生的图像的平滑度和分辨率。 几个其它技术存在，这提供的优点和缺点的不同组合。从样品的黑体辐射的直接红外成像是简单并具有几微米的空间分辨率，但是当样品是显著是唯一有效的LY高于室温。扫描探针显微镜热技术（例如扫描显微镜热电偶或开尔文探针显微镜）提供极好的灵敏度和空间分辨率，但是具有缓慢图像采集时间，必然由尖端的扫描速度，以及需要高度复杂的设备的限制。扫描激光或电子束扫描热显微镜措施时，一个调制光束跨过的电流偏置装置6，7，18的表面光栅化的电压扰动。这提供了优异的灵敏度，并且是比扫描探针技术稍快，但再次需要高度复杂的设备，并且还给出了样品的温度的间接，定性地图。

<table><tbody><tr><td>Europium thenoyltrifluoroacetonate powder</td><td>Sigma-Aldrich</td><td>176494-1G</td><td>Also known as Europium tris[3-(trifluoromethylhydroxymethylene)-(+)-camphorate]</td></tr><tr><td>Mercury short-arc lamp with flexible light guide</td><td>Lumen Dynamics</td><td>X-Cite Exacte</td><td>Light source includes internal iris and photosensor for output intensity feedback.</td></tr><tr><td>Peltier-cooled CCD camera</td><td>Princeton Instruments</td><td>PIXIS 1024</td><td>1,024 x 1,024 pixels, 16-bit resolution</td></tr><tr><td>610 nm band-pass filter</td><td>Edmund Optics</td><td>65-164</td><td>Passband has CWL 610&amp;nbsp;nm, FWHM 10 nm</td></tr><tr><td>500 nm short-pass filter</td><td>Edmund Optics</td><td>84-706</td><td>OD4 in stopband</td></tr><tr><td>Helium flow cryostat with optical window</td><td>Oxford Instruments</td><td>MicrostatHe2</td><td></td></tr><tr><td>high vacuum grease</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Digital Current source</td><td>Keithley</td><td>Model 2400</td><td>Computer-controllable current &amp;amp; voltage source</td></tr><tr><td>Digital Voltmeter</td><td>Hewlett-Packard&amp;nbsp;</td><td>Model 34420A</td><td>Digital Nanovoltmeter now available as Agilent Model 34420A</td></tr></tbody></table>

high-resolution thermal micro-imaging using europium chelate luminescent coatings

当偏置到它们的典型的操作条件微型电子设备通常经历显著自加热。本文介绍了一种方便的光学显微成像技术，其可被用于映射和量化这种行为。铕thenoyltrifluoroacetonate（EuTFC）具有612纳米的发光线，其活化效率随着温度的升高强烈下降，由于T中的Eu 3+离子和有机螯合化合物之间依赖性相互作用。这种材料可以通过在真空中升华热可以容易地涂布到样品表面。当涂层被激发紫外线（337纳米）的纳米612发光响应的光学显微图像可直接被转换成地图上的样品表面温度。这种技术提供（约1微米）的仅由光学显微镜有限的空间分辨率和时间分辨率由所采用的照相机的速度的限制。它提供的只是额外的好处需要相对简单的和非专用设备，并给样品温度的定量探测器。

用于进行该实验在低温浴温度下典型的测量配置的示例被示出在图1a中 ，而612纳米的发光响应强度与温度的典型曲线图1b绘制。 图2示出了的Bi 2的Sr 2 CaCu 2 O 8的THz源，其由堆叠的"固有"约瑟夫逊结的"台面"的尺寸为300×60×0.83微米的自加热的典型的热图像的?...

Watch this Scientific Journal Video about High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings at JoVE.com

High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings

铕thenoyltrifluoroacetonate（EuTFC）具有在612纳米，其激活效率随温度强烈降低的光致发光线。如果涂有这种材料的薄膜的样品是微成像时，612纳米的发光响应强度可以被转换成样品表面温度的直接映射。

高分辨率热显微成像采用铕螯合物的荧光涂料

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient ...

high-resolution-thermal-micro-imaging-using-europium-chelate

Universidad San Sebastian

Research

JoVE Journal

Engineering

7.7K 次观看.  Argonne National Laboratory. 铕thenoyltrifluoroacetonate（EuTFC）具有在612纳米，其激活效率随温度强烈降低的光致发光线。如果涂有这种材料的薄膜的样品是微成像时，612纳米的发光响应强度可以被转换成样品表面温度的直接映射。 

视频: High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

免疫与感染

Subsurface Defect Localization by Structured Heating Using Laser Projected Photothermal Thermography

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering thermal wave fields that are disturbed by the defect. This effect is measured and used to locate the defect. We form the destructively interfering wave fields by using a modified projector. The original light engine of the projector is replaced with a fiber-coupled high-power diode laser. Its beam is shaped and aligned to the projector&#39;s spatial light modulator and optimized for optimal optical throughput and homogeneous projection by first characterizing the beam profile, and, second, correcting it mechanically and numerically. A high-performance infrared (IR) camera is set up according to the tight geometrical situation (including corrections of the geometrical image distortions) and the requirement to detect weak temperature oscillations at the sample surface. Data acquisition can be performed once a synchronization between the individual thermal wave field sources, the scanning stage, and the IR camera is established by using a dedicated experimental setup which needs to be tuned to the specific material being investigated. During data post-processing, the relevant information on the presence of a defect below the surface of the sample is extracted. It is retrieved from the oscillating part of the acquired thermal radiation coming from the so-called depletion line of the sample surface. The exact location of the defect is deduced from the analysis of the spatial-temporal shape of these oscillations in a final step. The method is reference-free and very sensitive to changes within the thermal wave field. So far, the method has been tested with steel samples but is applicable to different materials as well, in particular to temperature sensitive materials.

This method aims at locating vertical subsurface defects. Here, we couple a laser with a spatial light modulator and trigger its video input to heat a sample surface deterministically with two anti-phased modulated lines while acquiring highly resolved thermal images. The defect position is retrieved from evaluating thermal wave interference minima.

使用激光投射光热法构造加热的地下缺陷定位

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering ...

工程学

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

生物化学

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs O2 sensitive luminescent sensor foils (planar optodes) in combination with a specialized camera system for imaging luminescence lifetime in the frequency-domain. Planar optodes are prepared by dissolving the O2-sensitive indicator dye in a polymer and spreading the mixture on a solid support in a defined thickness via knife coating. After evaporation of the solvent, the planar optode is placed in close contact with the sample of interest - here demonstrated with the roots of the aquatic plant Littorella uniflora. The O2 concentration-dependent change in the luminescence lifetime of the indicator dye within the planar optode is imaged via the backside of the transparent carrier foil and aquarium wall using a special camera. This camera measures the luminescence lifetime (&#181;s) via a shift in phase angle between a modulated excitation signal and emission signal. This method is superior to luminescence intensity imaging methods, as the signal is independent of the dye concentration or intensity of the excitation source, and solely relies on the luminescence decay time, which is an intrinsically referenced parameter. Consequently, an additional reference dye or other means of referencing are not needed. We demonstrate the use of the system for macroscopic O2 imaging of plant rhizospheres, but the camera system can also easily be coupled to a microscope.

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe the use of a novel, frequency-domain luminescence lifetime camera for mapping 2D O2 distributions with optical sensor foils. The camera system and image analysis procedures are described along with the preparation, calibration and application of sensor foils for visualizing the O2 microenvironment in the rhizosphere of aquatic plants.

使用基于频率域的摄像机系统的 O2的发光寿命成像

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs ...

化学

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

指南结构照明显微镜TIRF在高速，多种颜色

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Hyperspectral Imaging as a Tool to Study Optical Anisotropy in Lanthanide-Based Molecular Single Crystals

In this work, we describe a protocol for a novel application of hyperspectral imaging (HSI) in the analysis of luminescent lanthanide (Ln3+)-based molecular single crystals. As representative example, we chose a single crystal of the heterodinuclear Ln-based complex [TbEu(bpm)(tfaa)6] (bpm=2,2&#8217;-bipyrimidine, tfaa&#8211; =1,1,1-trifluoroacetylacetonate) exhibiting bright visible emission under UV excitation. HSI is an emerging technique that combines 2-dimensional spatial imaging of a luminescent structure with spectral information from each pixel of the obtained image. Specifically, HSI on single crystals of the [Tb-Eu] complex provided local spectral information unveiling variation of the luminescence intensity at different points along the studied crystals. These changes were attributed to the optical anisotropy present in the crystal, which results from the different molecular packing of Ln3+ ions in each one of the directions of the crystal structure. The HSI herein described is an example of the suitability of such technique for spectro-spatial investigations of molecular materials. Yet, importantly, this protocol can be easily extended for other types of luminescent materials (such as micron-sized molecular crystals, inorganic microparticles, nanoparticles in biological tissues, or labelled cells, among others), opening many possibilities for deeper investigation of structure-property relationships. Ultimately, such investigations will provide knowledge to be leveraged into the engineering of advanced materials for a wide range of applications from bioimaging to technological applications, such as waveguides or optoelectronic devices.

Here, we present a protocol to obtain luminescent hyperspectral imaging data and to analyze optical anisotropy features of lanthanide-based single crystals using a Hyperspectral Imaging System.

高光谱成像作为研究基于兰塔尼的分子单晶光学自给性的工具

In this work, we describe a protocol for a novel application of hyperspectral imaging (HSI) in the analysis of luminescent lanthanide (Ln3+)-based ...

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will allow successful eradication with antibiotics without an extra cost for a second surgery. Herein, we describe XELCI as a technique with high X-ray resolution, implant specificity, and chemical sensitivity to noninvasively image chemical concentrations near the surface of implanted medical devices. The devices are coated with chemically reporting surfaces. This chemically responsive surface consists of two layers coated on an implantable medical device; a pH-sensitive layer (bromothymol blue or bromocresol green incorporated hydrogel) which is coated over a red-light emitting scintillator (Gd2O2S: Eu) layer for monitoring. A focused X-ray beam irradiates a spot on the implant, and the red light generated by the scintillator (with 620 nm and 700 nm peaks) is transmitted through the sensing layer which alters the spectral ratio depending on the pH. An image is generated by scanning the X-ray beam across the implant and measuring the spectral ratio of light passing through the tissue point-by-point. We used this imaging technique for monitoring implant-associated infections previously on the bone surface of the femur with a modified implantable plate sensor. Now we are studying pH changes that occur from tibial intramedullary rod infections. Two different types of intramedullary rod designs are used in pre-pilot rabbit studies, and we learned that the XELCI technique could be used to monitor any chemical changes that occur not only on the bone surface but also inside the bone. Thus, this enables noninvasive, high spatial resolution, low background local pH imaging to study implant-associated infection biochemistry.

Here, we present a protocol for high-resolution optical detection of chemical information around implanted medical devices with X-ray excited luminescence chemical imaging (XELCI). This novel imaging technique is developed in our lab which enables studying implant-associated infection biochemistry.

通过组织的X射线激发发光化学成像对植入物相关感染进行高空间分辨率化学成像

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

构建用于扩展视场单分子成像的高倾角扫描磁贴显微镜的指南

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

高分辨率热显微成像采用铕螯合物的荧光涂料

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高分辨率光纤内窥镜为原位细胞成像

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

使用激光投射光热法构造加热的地下缺陷定位

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

使用基于频率域的摄像机系统的 O₂的发光寿命成像

指南结构照明显微镜TIRF在高速，多种颜色

高光谱成像作为研究基于兰塔尼的分子单晶光学自给性的工具

通过组织的X射线激发发光化学成像对植入物相关感染进行高空间分辨率化学成像

构建用于扩展视场单分子成像的高倾角扫描磁贴显微镜的指南

2维直接随机光学重建显微成像中间丝和微管

高分辨率热显微成像采用铕螯合物的荧光涂料

摘要

探索更多视频

此视频中的章节

高分辨率光纤内窥镜为原位细胞成像

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

使用激光投射光热法构造加热的地下缺陷定位

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

使用基于频率域的摄像机系统的 O2的发光寿命成像

指南结构照明显微镜TIRF在高速，多种颜色

高光谱成像作为研究基于兰塔尼的分子单晶光学自给性的工具

通过组织的X射线激发发光化学成像对植入物相关感染进行高空间分辨率化学成像

构建用于扩展视场单分子成像的高倾角扫描磁贴显微镜的指南

2维直接随机光学重建显微成像中间丝和微管

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

使用基于频率域的摄像机系统的 O₂的发光寿命成像