Name: using synchrotron radiation microtomography to investigate multi-scale three-dimensional microelectronic packages
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能源合同下的DE-AC52-07NA27344美国能源部劳伦斯·利弗莫尔国家实验室的主持下进行这一工作的一部分LLNL。英特尔公司笔者想从英特尔公司感谢刘毗临，两壶，威廉·哈蒙德和卡洛斯Orduno一些数据收集和有益的讨论。先进光源是由合同号DE-AC02-05CH11231主任，科学办公室，基础能源科学办公室，美国能源署的支持。

注:下述协​​议的细节是专门为工作在光束线8.3.2在ALS大学伯克利分校，加州写调整可能需要其他同步加速器设施，可以在全球各地找到了工作。适当的安全和辐射培训需要运行这些设施和训练的指导方针实验可以在每个单独的同步工具的网站上找到。任何更改或更新的断层协议（ALS，LBNL，伯克利，CA）可以在光束线手册15上找到。在断层扫描过程的细节可以参考16中找到。束线的科学家都可以回答任何问题，并将促进实验装置。 步骤1.在光束线执行断层扫描8.3.2（ALS，LBNL） <ol><li>通过将其安装在设计，以适应在束线的旋转载物台的样品夹持器准备为扫描样品。对于样品没有一个卡斯特OM接口，坚持样品后或钻夹头与粘土或蜡。 注意:在此研究中扫描的样品的微电子封装，为16×16毫米和高度仅为〜3毫米。为了适应整个包在视样品安装水平利用在束线提供粘土的字段。 <ol><li>对准样品，以保证当它旋转180度它保持在视野内。在厨内的旋转台装样前存在被用于对准样品的脱机模拟旋转台。旋转中心的目视检查是通常的取向就足够了。 </li><li>安装附加到厨内的样品支架的样品。一旦样品已经被安装在笼，两个正交定心电机使样品的位置相对于旋转的中心。 注意:有时需要样品制备提前实验时间，以确保该样品尺寸为所需的分辨率正确。例如，某些16 X 16 mm的微电子封装的切片成小块为进一步高分辨率扫描。可使用表1和2来确定样本大小。 </li></ol></li><li>选择用于基于感兴趣的样本大小和特征尺寸的扫描倍率。光束线8.3.2有几个镜头可供选择能产生与0.35的范围内的像素大小的图片 - 9微米。根据不同的放大倍数，样品必须是适当的横截面面积的，作为视场随放大率而减小。 <ol><li>因为这里扫描样品22.6毫米最长方向，选择1X透镜与PCO.4,000，如示于表1和2中，这种组合给出的视图最大样本场。所得像素尺寸为8.7微米。 </li></ol></li><li>设置x射线能量或切换到polychromatic光束使用光束线控制计算机。在束线8.3.2的X射线能量范围是连续的从4-80千电子伏，但多层单色安装限制了能量范围来〜7-43千电子伏，而峰值通量在约12千电子伏时发生。为了获得最佳的图像质量，立足于一个目标〜30％的传输，可在数据采集计算机上测量的能量选择。在提高能源一般情况下，传输％增加。 <ol><li>用于微电子封装，由于封装的厚度和材料"中选择白色"的光。 注:光束线8.3.2手动都有详细的步骤之间的"白光"和单色模式的变更。 </li><li>当使用"白"光模式中，为了筛选出低能量的X射线添加管线2-4金属铝和铜的过滤器与X射线束。对于此示例，使用2个铜板与〜1.2mm的总厚度。 </li><li>通过恬的样本计算提前传输e。通过使用: http://henke.lbl.gov/optical_constants/filter2.html或http://www.nist.gov/pml/data/xraycoef/或http://11bm.xray.aps.anl.gov/absorb/absorb .PHP。例如，输入为样本将输出表示百分透射率作为能量范围的函数的曲线图的化学式和估计的厚度。 </li></ol></li><li>验证旋转舞台的中心与​​相机的中心对齐。检查该样本是通过使用光束线控制计算机上的软件180度对准旋转，并通过查看在计算机上的X光片目视观察在样品的位置的变化。控制对齐改变同一台计算机上。当样品的取向是关闭的足够使样品的区域中的样品在旋转过程中离开的视场图像质量劣化。 </li><li>手动设置样品检测器距离的扫描。该相机是一个平移的阶段，可以水平移动所使用改变样品检测器距离。当距离增加相衬贡献也增加。相作用是有益的，以更容易地图像细微裂缝和边缘，而且还引起其他"光环效应"伪像通常是不希望的。 </li><li>验证束线对齐。检查图像的焦点，并在必要调整聚焦马达。确认该像素大小校准是通过移动样品一预定量和测量样品移动到计算微米/像素的像素的数量是否正确。该体素尺寸将取决于实验设置发生变化。 <ol><li>检查作为图像水平移动，图像配轨道沿着一个恒定的像素水平地，如果没有，让他们做调整摄像机俯仰电机。此对齐旋转轴，使得它平行于像素列，这是以后由重建算法假定对齐。 </li></ol></li><li>选择一个曝光时间为每个X光片。范围为曝光时间为1-1,500毫秒和选择取决于扫描能量和分辨率（它决定每个分辨率元素的观察到的通量）。在选定的时间应该提供最快的扫描时间与更多数量的扫描，因此最好信噪比之间的折衷。 <ol><li>对于微电子封装，使用每个曝光100毫秒的样本扫描时间。 注意:请确保没有饱和的像素或至少比该控制系统被设置为在转换后的规模显示摄像机数量，使每个相机的最大数量为65,535的100推荐的指标少。 </li></ol></li><li>使用数据采集计算机设置扫描参数。 <ol><li>输入所需的角度范围，和图像的数量以收集在该范围。的多个角度中选择的扫描时间越长，以及较大的数据集的大小。角度常见的数字是513，1025，和2,049超过0-180度的范围内。在这项研究中，使用1025角度数据采集过程中超过180度。 </li><li>选择扫描模式。扫描模式下的两个选项是:1）正常，2）连续断层。优选的连续模式，因为它会导致在最短扫描时间，〜3分钟。在这种模式下，为图像被收集的旋转阶段连续地移动。在正常模式下，旋转台停在每个角度，然后图像被收集。 </li><li>指定亮和暗场图像数量。明亮的暗场图像是必要的执行重建。对于暗场图像的快门关闭并且对于明场或背景的图像的样品移出视场的。验证该样品被转换得足够远，使得它未在亮场图像呈现，以避免在重建图像大的缺陷。在这里，获得15暗场图像和15亮场图像。 </li><li>确定是否贴砖是必要的。如果样品比外地高ÒF观看有一个平铺选项，它会扫描样品，然后直到整个样本捕获垂直翻译。 </li></ol></li><li>数据采集​​计算机上执行运行扫描。扫描将根据所输入的设置自动运行。 </li></ol>步骤2.表演断层数据处理<ol><li>将数据传送到一个​​分析计算机可在束线来执行使用束线协议的数据集的重建和过滤。重构可以运行独立于数据采集。 注:数据自动转移到NERSC，高性能计算机，它是处理和重建。用户可以在NERSC注册一个帐户spot.nersc.gov通过SPOT套房门户网站来访问他们的数据。该门户网站还处于发展模式，因此很多用户喜欢有超过重建参数，在这种情况下，他们按照其余步骤更多的控制。 </li><li> Reconst构作以下步骤将RAW图像:1）图像正常化，2）创建正弦图的叠加，3）申请环去除/过滤器，以及4）执行平行束重建。重建是基于滤波反投影算法。重建过程的结果，包含在每个像素构成的样品体积的位置和强度信息TIFF图像。整个过程的示意图示于图2。 <ol><li>要访问插件开始斐济（这是斐济的缩写，是刚刚ImageJ的），然后选择菜单→插件→ALSmicroCT NormalizeStack832newnaming如下图所示。在ALS设备用户可以使用ImageJ的/斐济，它集成了旨在简化重建过程中几个软件包自定义插件执行整个重建进程。 注:斐济和插件可用于多光束线8.3.2分析计算机使用。 </li><li>一旦斐济对话框打开如下所示，选择用于重建原始文件。原材料，明亮，和深色图像的堆栈现在应该被加载。 </li><li>通过点击"旋转检测中心"，然后以可视化的重建图像选择"预览重建"查找旋转中心。为旋转中心的值，也可以手动输入和预览。 </li><li>使用这个接口有改变环除去参数的选项，图像（8，16或32位），象素范围的类型，图像的旋转角度，并限定裁剪区域。每一个新的参数集可以使用&#39;预览重建"按钮被可视化。 </li><li>一旦参数被选择，通过选择"运行"重建图像的整个堆叠。所有后续数据文件可以在指定的"输出目录"中找到，默认的目录将在原始数据文件夹中的输出文件。 </li></ol></li><li>从断层的访问原始数据通过进入网站http://spot.nersc.gov/，这是NERSC（LBNL超级计算机）服务器通过SPOT门户从任何计算机罐。 注意:每个单独的研究人员必须有自己的NERSC帐号来访问他们的特定数据集。用户可以设置在https://nim.nersc.gov/nersc_account_request.php一个帐户。在束线，各研究组被分配一个束线的帐户。此帐户用于访问束线计算机上，并且也可用于直接从使用的Globus在线束线服务器访问数据。 </li><li>通过加载2D的堆叠可视化3D和2D数据重建的图像转化成任何3D分析软件。这里介绍的样品和图像使用Avizo软件来执行分析和可视化，这可在任何的束线8.3.2分析计算机来束线用户。 </li><li>一组数据被上传到可视化软件后进行进一步的数据分析以获得关于特定FE量化信​​息样品内atures。常数据集下采样以便减少所述输出数据的大小。然而，这可以增加像素尺寸缩小的保真度，但平滑图像视图，便于分割。 <ol><li>由阈值2D的叠层的直方图重建的切片和分配一个新的像素值落入规定范围内的像素选择感兴趣段特征。 </li><li>可视化分段体积和表面。一旦特征被分割他们正在使用Avizo或首选的可视化软件观看3D的。这允许特定功能的三维表面渲染，就像在某个感兴趣区域的焊球。 </li><li>量化在样品的特征， 即 ，裂纹尺寸，通孔，孔隙率，缺陷等 。一旦感兴趣的特征是通过识别诸如或裂纹，特征可以分段和裂缝宽度，长度的体积信息，通过体积，孔隙率分布可以通过评估tomogr量化aphic数据集。 </li><li>创建影片表示不同的取向样品的样品。电影1示出的用于在水平方向成像的微电子封装的不同的横截面视图和体绘制视图的示例。 </li></ol></li></ol>

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作者什么都没有透露。

所有的在协议部分中描述的步骤是获得多尺度和多材料样品的高分辨率图像的关键。其中最重要的步骤是将样品放置和光学，这对获得可用于定量质量的图像的重要的聚焦。具体地，即使样品的轻微运动将导致伪像的重建图像中和散焦将导致分辨率的恶化。以避免图像质量问题它重建一个测试图像，从而可以同时进行，而下一个样品扫描是重要的。这将有助于识别可能在扫描设置期间发生的任何?...

在微电子领域中，如在许多其它领域，表征样品时在微米尺度的非破坏性评价是必要的。专门用于微电子工业中有探测三维微电子封装，含有多层次，多材料，并且在热学，电学，和机械应力的组件的标识中的包的故障的兴趣。在世界各地同步辐射设施已指定了用于微电子封装的失效分析断层扫描和衍射光束线。其中一些例子是成像造成电1-3空洞形成，在评估锡须生长4,5 ​​机制， 在过冷和锡的各向异性的热膨胀和金属间化合物（法团校董会）6,7的实地观测， 在原位观测凝固并形成IMC 8-10，各向异性的机械行为，锡的再结晶和无铅焊料10，空隙在倒装芯片凸点，并在银nanoink烧结11的实地观测。所有这些研究都进一步推进了微电子工业的理解和组件的开发。然而，许多这些研究都集中在包内的小区域。更多信息，可从测试和使用高分辨率SRμT为了进一步发展自己的特性全尺寸封装中收集。 所生产的电子产品的现在包含互连的多层。这些包和设备正变得越来越多的复合体用于非破坏性评价关于故障分析，质量控制，可靠性风险评估，和发展的三维溶液呼叫。某些缺陷需要，可以检测出特征小于5μm的大小，其中包括空隙和裂缝铜ス内部形成技术bstrate通路，识别非接触式开放和nonwet焊盘在多层包装12中 ，定位和定量在球栅阵列（BGA）和C4焊点空隙。在衬底组件过程中，这些类型的缺陷必须确定并广泛地监测，以避免不必要的故障。 使用基于实验室的来源，也称为桌面目前CT系统中，能够提供高达〜1微米空间分辨率，和被用来隔离在与有希望的结果的多级封装的故障。然而，相对于SRμT设置13,14时桌面CT系统有一定的局限性。桌面系统仅限于成像的材料，因为它们通常仅包含一个或两个x射线源光谱一定的密度范围内。此外通量时间（TPT）保持长期为需要几个小时的数据采集时间每兴趣1-2毫米2区域，该区域CA传统的桌面CT系统n个极限其实用性;例如，穿硅通孔（TSV），BGA的或C4接头通常需要在样品中采集感兴趣视图（FOV）或区域的多个场的分辨率高，导致在分析失败的8-12小时总TPT，这是一个显示塞何时多个样品必须分析传统的桌面CT系统。同步加速器辐射提供高得多的通量和亮度比传统的x射线源，从而导致更快的数据采集时间为感兴趣的给定区域。虽然SRμT确实允许用于相对于类型的可以成像和样品体积的材料更大的灵活性，但它确实有局限性，这是特定于同​​步加速器源和使用的设定，具体而言最大可接受厚度和样品大小。为ALS的SRμT设置可以被成像的最大截面积为&lt;36×36毫米，厚度是由能量范围和磁通可用限制，并且是材料Specific。 本研究用于演示SRμT可以如何被用于图像中的封装（SIP）的带有使用高分辨率和低TPT（3-20分钟）的整个多级系统中检查三维半导体封装。在比较桌面CT对同步加速器源CT的更多细节可以在参考文献13,14找到。 实验概述和光束线8.3.2描述: 有可为世界各地的断层实验同步加速器设施;大多数这些设施需要提交所在的实验者描述了实验，以及其科学影响力的提议。这里描述的实验都在ALS劳伦斯伯克利国家实验室（LBNL）的光束线8.3.2执行。对于此束线有两种能量模式选择:1）在能量范围〜7-43千电子伏或2）多色"白"光，其中整个availa单色扫描高密度材料时，竹叶提取能量谱被使用。期间在束线8.3.2一个典型的扫描一个样本被安装在一旋转台，其中x射线穿透样品，然后将衰减的X射线通过闪烁器转换成可见光，由透镜放大，然后投射到CCD记录。这是当样品从0到180°产生的图像的堆叠是重建以获得具有微米分辨率的样品的3D视图旋转完成。所得断层数据集大小从〜3-20千兆根据扫描参数的范围内。 图1示出了样品被扫描的笼的示意图。 这里提出了以下方案描述了实验装置，数据采集，和用于成像的整个微电子封装所需的处理步骤，但该步骤可被修改，以图像的各种样品。修改取决于样品的大小，密度，几何形状，和特征的兴趣。 表1和2本可在束线8.3.2分辨率和样本大小的组合（ALS，LBNL，伯克利，CA）中。对于这里研究的微电子封装的样品用的多色（"白"）的光束，这是由于厚度和样品的组分的高密度选成像。将样品安装在水平方向上的卡盘架，该取向允许整个样本以适应光束，这是具有〜4个毫米，〜40毫米宽的高度并行的高度内，因此只需要一个扫描捕获整个样本。

<table><tbody><tr><td>Beamline 8.3.2</td><td>Advanced Light Source, Berkeley, CA, USA</td><td></td><td>http://microct.lbl.gov/</td></tr></tbody></table>

using synchrotron radiation microtomography to investigate multi-scale three-dimensional microelectronic packages

同步辐射的微断层摄影术（SRμT）是一个非破坏性的三维（3D）成像技术，提供了高通量为高空间分辨率的快速数据采集时间。在电子行业中有三维微电子封装，其中许多含有高密度互连的多个层面进行故障分析的浓厚兴趣。常在断层有图像分辨率和可成像的样品的体积之间的折衷。这种反比关系限制常规计算机断层扫描（CT）系统的实用性，因为微电子封装的横截面面积100-3,600毫米2常大，但对微米尺度的重要功能。在先进光源（ALS），在加州伯克利USA，微断层扫描光束线有一个设置是适应性强，可以根据样本的属性， 也就是说，密度，厚度等 ，最大允许的36×36毫米能够截面。这样的设置也有成为全能量范围〜7-43千电子伏无论是单色或使用多色束白光模式下最大磁通操作选项。这里呈现的是一个包中采取的图像的整个16×16 mm系统的实验步骤的细节，以便获得系统的3D图像具有8.7微米的所有的空间分辨率小于3分钟的扫描时间范围内。还示出了从不同的方向和一个切片包更高分辨率成像扫描包的结果。对比度的常规CT系统将需要数小时具有潜在的较差的分辨率来记录数据。事实上，使用同步加速器辐射断层设置时场的视图可以通过时间的比例要高得多。下面的实验装置的说明可以实施并适于使用与许多其它多材料。

使用断层摄影捕获的图像的发生是由于X射线在焊料互连，金属迹线和其他材料在微电子封装的不同衰减长度的函数，并且这些多材料的厚度的差的吸收。该SIP封装模具包括连接到直径约80微米的第一级互连（FLI）倒装芯片C4焊球陶瓷基板硅的;中级互连（MLI）约350微米的焊球该衬底连接到FR4环氧电路板;以及第二级互连（SLI）的在电路板的背面侧大约650微米的BGA焊球图2示出了当它被放置...

Watch this Scientific Journal Video about Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages at JoVE.com

Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages

在这项研究中同步加速器辐射微断层摄影术中，非破坏性的三维成像技术中，用于研究整个微电子封装为16×16毫米的截面积。由于同步的高通量和亮度样品成像，在短短3分钟，8.7微米的空间分辨率。

利用同步辐射微断层调查的多尺度三维微电子封装

Synchrotron radiation micro-tomography (SR&#181;T) is a non-destructive three-dimensional (3D) imaging technique that offers high flux for fast data ...

using-synchrotron-radiation-microtomography-to-investigate-multi

Research

JoVE Journal

Engineering

10.0K 次观看.  Lawrence Livermore National Laboratory. 在这项研究中同步加速器辐射微断层摄影术中，非破坏性的三维成像技术中，用于研究整个微电子封装为16×16毫米的截面积。由于同步的高通量和亮度样品成像，在短短3分钟，8.7微米的空间分辨率。 

视频: Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.
Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of &gt; 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.
Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.

The use of energy dispersive X-ray tomography in the scanning transmission electron microscope to characterize elemental distributions within single nanoparticles in three dimensions is described.

能量色散X射线断层摄影术对个人纳米3D元素映射

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial ...

科研

工程学

Failure Analysis of Batteries Using Synchrotron-based Hard X-ray Microtomography

Imaging morphological changes that occur during the lifetime of rechargeable batteries is necessary to understand how these devices fail. Since the advent of lithium-ion batteries, researchers have known that the lithium metal anode has the highest theoretical energy density of any anode material. However, rechargeable batteries containing a lithium metal anode are not widely used in consumer products because the growth of lithium dendrites from the anode upon charging of the battery causes premature cell failure by short circuit. Lithium dendrites can also form in commercial lithium-ion batteries with graphite anodes if they are improperly charged. We demonstrate that lithium dendrite growth can be studied using synchrotron-based hard X-ray microtomography. This non-destructive imaging technique allows researchers to study the growth of lithium dendrites, in addition to other morphological changes inside batteries, and subsequently develop methods to extend battery life.

Synchrotron-based hard X-ray microtomography is used to image the electrochemical growth of dendrites from a lithium metal electrode through a solid polymer electrolyte membrane.

电池的使用同步为基础的硬X射线微断层故障分析

Imaging morphological changes that occur during the lifetime of rechargeable batteries is necessary to understand how these devices fail. Since the advent ...

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

lensfree芯片断层用人多角度的光照和像素超高分辨率的显微镜

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

生物工程

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

生物学

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

发育生物学

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

冷冻电子断层扫描远程数据收集和断层扫描平均

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

利用同步辐射微断层调查的多尺度三维微电子封装

摘要

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此视频中的章节

能量色散X射线断层摄影术对个人纳米3D元素映射

电池的使用同步为基础的硬X射线微断层故障分析

lensfree芯片断层用人多角度的光照和像素超高分辨率的显微镜

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

相关显微镜的动力相互作用的三维结构分析

使用微计算断层扫描的果蝇黑色素加斯特全动物成像

准备厚生物样品的观察扫描透射电子断层扫描

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像

冷冻电子断层扫描远程数据收集和断层扫描平均

利用同步辐射微断层调查的多尺度三维微电子封装

摘要

探索更多视频

此视频中的章节

能量色散X射线断层摄影术对个人纳米3D元素映射

电池的使用同步为基础的硬X射线微断层故障分析

lensfree芯片断层用人多角度的光照和像素超高分辨率的显微镜

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

相关显微镜的动力相互作用的三维结构分析

使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

准备厚生物样品的观察扫描透射电子断层扫描

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像

冷冻电子断层扫描远程数据收集和断层扫描平均

使用微计算断层扫描的果蝇黑色素加斯特全动物成像