Name: identification of metal oxide nanoparticles in histological samples by enhanced darkfield microscopy and hyperspectral mapping
Uploaded: 2015-12-08T14:00:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping at JoVE.com

The authors thank G&#252;nter Oberd&#246;rster, DVM, PhD and Alison Elder, PhD (University of Rochester) and Mary Frame, PhD (Stony Brook University) for animal research collaborations resulting in tissue samples for analysis. Additionally, the authors thank: Christina Rotondi (Albany Medical College Histology Core); Rani Sellers, DVM, PhD and Barbara Cannella, PhD (Albert Einstein College of Medicine Histology and Comparative Pathology Facility); Leonardo Bezerra and Ahlam Abuawad (Brenner research team members); and Leslie Krauss, Byron Cheatham and Elyse Johnson (CytoViva). This work was supported in part by CDC-NIOSH grant OH-009990-01A1 and the NanoHealth and Safety Center, New York State, awarded to S.B.

动物协议在调查人员的合作机构，州立大学石溪分校获得批准的机构动物护理和使用委员会。具体的材料和用于此造纸设备的列表可以在表1中找到。 组织1.样品制备<ol><li>准备暴露于金属氧化物纳米颗粒以用于组织学或免疫组织化学染色根据先前描述的方法21-23的动物组织。 </li></ol> 2.影像<ol><li>初始化显微镜 注意:对于该研究中，研究级显微镜下，配备一个高性能的暗视场光源，电动XY载物台控制器，14位深度高光谱照相机，和多个物镜（10X空气，40X空气，100X油浸） 。这里使用的系统具有64纳米的空间分辨率在100X（油​​浸）放大率。用于该螺栓冷凝器y具有两种克勒和临界照明的功能和重点的高准直光的上样倾斜的角度。 <ol><li>插入并打开光源，XY工作台控制器，光学照相机（暗场成像），高光谱相机和计算机系统。设置光源，以75％的电力;提高阶段最大高度;连接导光到冷凝器增强暗场成像，或在显微镜的背面为反射明成像准直仪。 注:光源设置为75％的电力，有整个视野，增强暗淡像素，同时仍然允许枯燥和明亮像素之间的反差之内的所有像素的充分，均匀的照明。 </li><li>提高冷凝器的工作位置。应用3-5滴A型浸没油对聚光透镜小心，避免任何气泡的形成。擦去油，并重新涂抹，如果气泡应该形成。 </li><li>放置幻灯片Øn为舞台。慢慢地将冷凝器，直到浸油，使与滑动接触。这将是明显的，通过照明的迅速增白环在油使得与滑动接触。 </li></ol></li><li>把10X目标到位。福克斯和检查通过目镜对准冷凝器。 <ol><li>通过粗目标调焦旋钮移动舞台上下，直到亮度最大化。 </li><li>移动冷凝器集中起来，并通过冷凝器调节旋钮，直至最大亮度被找到。尝试创建中的视场中的亮的中央点的可能。 </li><li>根据需要调整冷凝器调整旋钮，如果需要到中心亮点。 </li><li>使用细目标调焦旋钮使亮点成为关注的焦点。当改变目标，以获得不同的放大倍率，调整焦平面。当利用100X目标，应用一滴浸油过的盖玻片，以加强日Ë形象，避免损坏物镜。 </li></ol></li><li>捕捉图像<ol><li>使用级控制器找到感兴趣的区域。该区域将被实验的需要来确定。一个典型的指标将与周边地区的高对比度，为泛着金属氧化物纳米颗粒的地区经常出现比没有他们的区域更亮。 </li><li>使用细客观调焦旋钮，调整所必需的平衡照明的聚光焦点把该地区成为关注的焦点。视野内获得高对比度，良好定义的区域。 </li><li>确定哪些图像（光学摄像机）或datacubes（用于高光谱摄像机）将被捕获，以及以何种顺序。通常情况下，光学图像与10倍的空中目标，40X空中目标，并100X油浸物镜和相应的恒指datacube用100X油浸物镜获得。 <ol><li>打开光学成像软件。点击"设置 -＃8221;在菜单栏中。选择"图像捕捉按钮捕捉事件"。选择所存储的图像格式（TIFF用于此实验）;指定一个文件名;浏览并选择存储的图像文件夹;保持默认缩时;单击"确定"。 </li><li>选择创建最高对比度图像中的"曝光"菜单中的曝光设定（对于这项研究中，0.0％的水平，为3.0 dB的增益，和35毫秒的快门被使用）。 </li><li>通过单击菜单栏中的"图像"按钮捕获图像。通过改变显微镜物镜，为了提供上下文捕获几个低倍率暗场图像除了那些在高放大倍数。 注:在相同的放大倍数为将与超光谱照相机捕获任何datacubes捕捉光学图像，因为这些光学图像倾向于具有来看，更好的美学外观购买目视检查较大的场。重要的是，任何DATACubes稍后将待光谱分析使用一致的放大率，因为它有可能改变目标将改变显微镜光学器件的透射光谱，改变所捕获的光谱，并因此减少了光谱角映射器（SAM）中的精度。如果一个人datacube相比用不同的目标，得到另一datacube，在SAM功能可能无法正常工作。 </li></ol></li><li>通过重定向在显微镜的光源旋钮超光谱照相机选择高光谱照相机成像检测器。如果光被引向高光谱照相机，无图像将被显示在光学摄像机软件。最小化，但不要关闭光学成像软件窗口，为一个可能需要按步骤2.4.4规定。 </li></ol></li><li>捕获Datacubes <ol><li>打开收购恒指datacubes的高光谱成像软件。确保导光被引导到高光谱相机。 </li><li>打开"HYPerspectral显微镜"在菜单栏中选择"恒指显微镜控制"。 </li><li>设置物镜放大倍数和保存路径。确保命名鲜明所以没有覆盖发生的所有图像和文件。通过调整视图或行（使用720线为这项研究）的数量方面，有更多的时间需要捕获更大的领域显著量更改的区域捕获的设置。最后，设置曝光时间（0.25秒，这项研究）。离开一切为默认，然后单击"预览恒指"查看图像。 注意:显示强度图表显示HSI datacube将被记录在水平轴。纵轴表示将在HSI datacube被捕获的光谱的波长。将光标置于在对应于一个波长的光谱强度曲线的任何位置将导致在图像的所有点的强度，在该波长，被证明。 </li><l我&gt;焦点在此基础上预览，通过调整细目标重点锐化在这个形象的峰值。如果这是太困难，光学相机的焦距是相当相似，所以加载光学成像软件和滑杆转移到光学照相机，对焦，然后返回到高光谱成像软件和滑动杆返回到高光谱相机。 </li><li> ，或取消预览调整曝光时间调整在预览通过调节光源亮度，聚光焦点的强度。后者得到最佳的效果，反而增加曝光时间需要更长的图像。这样做的目的是为了更显著峰得足够大，但不超过最大强度为检测器;在这里，理想的范围内16000之间的1000和单位。 </li><li>点击"捕获"。该显微镜将要求带的暗电流图像。重定向上滑动杆或光圈（小心，以免打扰排列和任何其他光学焦点），并单击"确定"。恢复滑动条或光圈到正确的位置，然后再次单击"确定"形象出现提示时。影像可能需要长达30分钟，但大约5分钟的时间是比较典型的。更长的曝光时间导致更长的成像时间。进度指示器存在。要小心，不要打扰物理范围进度指示器完成之前。 </li><li>观察四个新的窗口，名称分别为:"可用波段列表"，"＃1zoom"，"＃1scroll"和"＃1 RGB带"。最大化"＃1RGB乐队"的窗口，因为这是datacube的所有引用。 </li><li>右键单击datacube并将其保存为TIFF，然后单击"确定"。如果完成了成像;收起所有的样品;清洁所有油暴露表面在水中的70％异丙醇;提高阶段最大高度;较低的冷凝器，以最小高度，然后按成非工作位置;关闭向下或拔出光源，工作台控制器，和摄像头。 </li></ol></li></ol> 3.创建参考光谱图书馆<ol><li>选型参考光谱<ol><li>选择已知含有的包含在同一矩阵作为实验样品感兴趣的材料，因为HSI光谱曲线是基质依赖性阳性对照。 <ol><li>在这项研究中，使用猪皮肤注射高剂量的金属氧化物纳米颗粒作为阳性对照用于比较的实验猪皮肤组织从一个局部暴露研究。生成的金属氧化物纳米颗粒在悬浮液中的光谱曲线和检查，发现是不恰当的阳性对照组织学实验样品由于不同的矩阵。 </li></ol></li><li>获得来自阳性对照一个datacube如步骤2.4所描述的，使用高光谱相机。 <ol><li>特别谨慎的时候本身拟合的强度，因为这是主度量通过其内部粒子过滤器将标识材料。上面的检测器（在此为16000单位）的饱和点的任何强度将导致无效的数据（参照步骤2.4.5）。 </li></ol></li><li>右键单击图像窗口，点击左侧的"Z-轮廓谱"。会出现一个弹出式光谱图的窗口。左键点击到感兴趣的datacube像素，特别是最亮的人，或那些可以自信地确定为代表感兴趣的材料。观察频谱轮廓窗口显示相关的频谱。注意到特别是他们的最低和最高值，其波长对应于它。 <ol><li>当通过点击感兴趣的非常明亮的相对于周围的组织，尤其是那些具有容易识别的颗粒，它们是区域勘测阳性对照样品，调查。这些颗粒是最有可能是脑膜感兴趣IALS，特别是在金属的情况下。 </li></ol></li><li>使用下的"颗粒分析"菜单中的"粒子过滤器"工具来确定存在于datacube颗粒。在新弹出的窗口中，应遵守"最高的光谱必须超过"。这将通过观测步骤3.1.3确定。设置此值，因此它比背景像素比感兴趣的材料高，但低。的"有效数据最大"是最大强度（这里，16000单位）。 <ol><li>离开其他参数为默认值，但通过调整"大小阈值"框排除基于大小的物体。保存该数据要么在这一点上，或运行分析后。对于该实验，使用以下参数:光谱最大值必须超过:5000;有效数据最大值为16000;大小阈值（像素）:400。一旦所有参数已经确定，单击"确定"。 </li></ol></li><li>观察所得图形以的细节在指定的强度阈值检测颗粒;这个数据可以导出。如果感兴趣的材料的特性最大反射率波长是已知的，选择那些有类似的"最大WL"值的颗粒;否则，单击"全选"。然后单击"导出"; "光谱库"。选择一个文件名，然后单击"确定"。 </li></ol></li><li>取出假阳性谱<ol><li>选择，将作为阴性对照的样品。这样的样品应已制备并以相同的方式处理所有的实验样品保存，由于没有纳米颗粒类似或相同的所关心的材料的有保证。 注意:重​​要的是，与阴性对照的矩阵是相同的实验样品的基质，如HSI光谱曲线是基质依赖性。在这项研究中，我们使用猪皮肤不暴露在金属氧化物纳米材料为负控制。 </li><li>如在步骤2.4.7所述，使用高光谱相机获取从阴性对照几个datacubes。中的至少一个是必需的，但更可以被捕获，以增加选择性（这是在事件尤其重要的是，某些污染物可能在参考光谱）。 </li><li>使用该高光谱成像软件来过滤收集在步骤3.1（阳性对照）针对每个datacube的光谱库在步骤捕获3.2.2（阴性对照）串联的指示，在下面的步骤: 注:保存所得，过滤光谱库到一个单独的文件，并作为用于感兴趣的材料的参考光谱库使用。 <ol><li>点击"过滤谱库"下的分析菜单，在主编程工具栏上。点击"打开"; "新建文件"，并选择在步骤3.1创建的光谱库。为阳性对照作为输入文件。点击"确定"。 </li><li>要使电子邮件xternal源，选择在光谱数据中的"形象"。保留下的工艺参数对话框的默认设置。选择一个输出名称对于筛选库（这应该是比原来的不同，或收集的原始光谱库将丢失）。点击"确定"。 </li><li>点击"打开"; "新建文件"，然后选择为阴性对照，出现提示时选择源图像的步骤3.2.2捕获的第一datacube。 注:该软件将分析光谱库，并删除每个光谱相匹配的阴性对照datacube的任何频谱。删除从选择标准的背景，从而减少潜在的假阳性。的摘要，将可用这样做时（注意，此概述并不自动保存）。 </li><li>如果额外的过滤需要，从步骤3.2.3.1重复，除非:3.2.3.2步，选择创建的最后一个过滤谱库（导致步骤3.2.3.3后联合。mpletes）;在步骤3.2.3.3，请从步骤3.2.2下一datacube。 注:校正和正火对于灯频谱可能是必要的，如果样本散射相当量的光（例如，碳纳米管）和/或如果当针对阴性对照过滤阳性对照光谱库零谱仍然存在。并没有出现我们研究这些情况，因此没有进行这种修正。 </li></ol></li></ol></li></ol> 4.图像分析<ol><li>光谱角制图<ol><li>打开来自谱菜单，映射方法的子菜单的"光谱角映射器"（SAM），利用超光谱成像软件一旦所有的实验datacubes已经获得以下步骤2.4。光谱角映射器通过几何分析变化强度作为波长的函数的光谱进行比较（即，它通过正常化它们的强度和比较所述角度重新比较两个谱quired追踪每个光谱的曲线图）24。 </li><li>随着datacube打开时，选择实验datacube在弹出窗口的名称，然后单击"确定"。如果没有文件名列出，单击"打开"; "新文件"，选择实验datacube，然后单击"确定"。 <ol><li>观察称为端元收集新的弹出窗口:SAM。点击"导入"，在菜单栏，然后​​选择"从光谱库文件"中选择。一个弹出窗口命名谱库输入文件将会出现。打开那在步骤3.2.3创建的参考谱库。并单击"确定"。注意命名为"输入谱库"的新的弹出窗口。 </li><li>点击"选择所有项目";单击"确定"右键点击"颜色"，然后选择"应用默认颜色为所有"。单击"全选"，其次是"应用"，然后选择输出文件名称的d单击"确定"。该光谱角映射器将需要几秒钟来分析和保存数据。 </li></ol></li><li>在打开的高光谱成像软件datacube。在图像窗口的叠加菜单中选择"分类"，然后定位到该文件名，然后单击"确定"。用户现在可以覆盖任何频谱从库看到他们映射到图像中。 </li><li>如果一个统一的配色方案需要，如其他软件更容易分析（如步骤4.2节选择在"交互类的工具"，通过在图像窗口的叠加菜单中打开，在选项菜单中的"合并类"选项）。 </li><li>突出显示所有的分类组合（通常，在一切，除了"机密"，"类合并成基地"的列表），然后从"基类"列表中的单个频谱，然后单击"确定"。该颜色将立即repres耳鼻喉科所有选中的光谱。 </li><li>点击所选择的颜色和所有匹配的频谱将在该颜色显示。 </li><li>为了获得二色图像，将显示在黑色背景上，cIick的"未分类"的色彩中，这是黑默认匹配光谱。观察二色的图像。这个步骤可以通过在"未归类"颜色盒再次单击颠倒。 </li><li>右键点击将图像保存为TIFF，包括任何覆盖当前活动。 注:对于未来的成像处理中，二色图像将被使用。 </li><li>从选项菜单中选择"级分配"中的"互动类工具"窗口来获取映射统计数据。这个数据代表了很多像素是如何映射的（未分类），有多少被确定为感兴趣的材料。注意，像素的数量不具有映射颗粒的数量相对应。这个信息是不会自动řecorded。 </li></ol></li><li>在高光谱粒子分析图图像<ol><li>打开在NIH ImageJ的软件图像。 </li><li>使用图像菜单，调整子菜单，"阈值"的功能。选择区分的所有其他材料感兴趣粒子参数。使用下面的阈值参数研究:默认情况下阈值法，色红，色彩空间HSB，检查深色背景复选框，选中通复选框色调，饱和度和亮度。 注意:这可能从案件情况显著改变。当分析映射datacube，这可以通过统一的所有相关类单一颜色和覆盖所有非保密颜色彩色产生图像（步骤4.1.4至4.1.8）之前被简化。 </li><li>使用分析菜单，"分析粒子"功能，这将检索的信息区域，意思是，最低和最高值。在这项研究中，使用此参数:尺寸0无穷远，圆0-1，什么都不显示，检查显示效果复选框。 </li><li>进行统计分析，此数据输出到另一个软件程序实现与位于类似对照样品中颗粒的数量和大小的统计比较。我们建议使用电子表格的ImageJ的数据分析。 4.2.4步后显示在结果表中的数据复制到电子表格。 MAX函数可用于在第一（无）柱来确定颗粒的数目，而平均函数可用于在区列，以确定粒子的平均尺寸。在未来，实验验证将继续执行在确定平均大小的已确立的标准尺寸（例如，TEM）调查此功能。 </li></ol></li></ol>

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The authors have nothing to disclose.

对于已经经过常规组织学染色，金属氧化物纳米粒子的识别和分析的组织样品可以通过EDFM，HSI映射和图像分析技术的组合来实现。虽然在用于组织学或免疫组织化学的样品制备的灵活性（例如，使用固定的或冷冻的组织;色斑的类型），但重要的是样品切片，以获得最佳的可视化的厚度为5-10微米。这里使用的样品是福尔马林固定和石蜡包埋，用旋转切片机切片至6微米厚之前，安装在玻璃显微镜载片上，用苏木精和曙红和盖上盖玻片。猪皮肤的组织从活体外皮肤渗透毒理学协作用于该研究。将组织暴露于金属氧化物纳米颗粒（氧化铝，二氧化硅，二氧化铈）的水悬浮液。检测感兴趣区域（S）的（高对比度elemeNTS）与EDFM是促进后续恒指映射和分析关键的第一步。阳性和阴性对照样品必须被成像和第一，以便创造一个光谱库，以供参考分析。从阳性对照所收集的光谱输出到阳性对照光谱库。然后，从阴性对照图像所有光谱是从阳性对照的光谱库中减去，以提高特异性（减少误报）。由此产生的过滤谱库被认为是服务于感兴趣的材料分析认为，RSL。所有的组织样本进行相同的成像过程和被映射对RSL。由此产生的图像将包含与目标在黑色的背景元素专用区。此图像然后可以用使用ImageJ其阈值和粒子分析功能，以获得每个视场映射粒子的面积进行分析。从ImageJ的获得的数值数据可以导出编到一个电子表格作进一步的分析。 考虑到，作为生物样品彼此本质上的不同，和染色方法可以通过EDFM和HSI影响可视化，曝光设置，应根据什么产生最佳的高对比度图像为样本的特定类型来确定是很重要的。虽然减少误报可以通过光谱库的过滤来实现，这是至关重要的，以获得已经避免污染与所关注的元件可靠阴性对照，作为相应于这种污染的光谱可能被从阳性对照的光谱库过滤，增加假阴性率。另外，光谱强度范围可检测与超光谱成像软件不能超过特定的软件的限制（对于本研究，这是16000单位）:产生​​菌具有大量的积累颗粒区域ectral强度以上的强度极限被排除的光谱库的，由于增加的假阴性的数量的风险。 虽然恒指系统比传统方法赋予许多优点，也有一些缺点和局限性考虑。之一是，大量收集可能需要大量的计算能力的光学数据。另一个原因是，恒指可能需要大量的时间，特别是在开始阶段，当正在创建的参考谱库。此外，成像时间可能要求每个图像采集几分钟的时间，使得它比简单的暗场成像速度较慢;然而，它仍然比通过电子显微镜进行采样制备和可视化更快。此外，复杂的系统可能导致多个特性谱，这需要高度专业化的控制的发展做出建立标准化，通用的基准库26困难。最后，高科技NIQUE结果在分辨率比探针 - 或电子显微镜技术，例如原子力显微镜或透射电子显微镜，可以分辨单个原子的低级。该技术的分辨率是有限的，因为它的光子性质，从而防止它从一个高精度工具，用于测量颗粒尺寸在纳米级或用于以亚微米级精确定位的材料。而该技术可能能够查明某些组织区室或细胞器内的颗粒大于1微米（如细胞核），小细胞器或特征是具有挑战性的准确形象化用这种方法。还值得注意的，因为它的空间分辨率，该方法不能单一纳米颗粒和附聚物11区分。 其他考虑因素包括:某些材料（如贵金属）具有非常高的反射率和不同的光谱图，它可以使它们更容易ANALY泽而与此工具的光谱图。其它如研究本研究中的半金属的氧化物和碳基纳米材料24，27，可以是更具挑战性，因为它们的元素组成，形状和视矩阵。在经Mercer等人的2鼠吸入研究，一个类似的系统，以一个在这项研究中使用的，使用，以找到碳纳米管在肺和基于其与周围组织显着高的对比度次级器官。然而，高光谱制图未在任一研究表明，可能是因为碳纤维的独特形状为足够性状进行识别。另一个要考虑的是关于具体组织:自沉积纳米粒子通过正常的生物物理过程感兴趣的特定器官是不可预测的（并且经常自己研究的课题），确定适用的阳性对照的是很困难的，需要考虑的是如何生成的控制米的飞行影响感兴趣的材料的状态。例如，如果一个光谱库是从感兴趣原始纳米颗粒创建时，它可能难以映射库的那些相同的纳米颗粒在组织或细胞中由于改变光谱从变更粒子（如所导致，由于改变pH值，溶解，结块，蛋白质结合）和整体微环境或基质。最后，该技术在其半定量性质的限制:它只能是定量为相似的分辨率，这意味着它不能容易地用于执行任务，例如表征材料的总器官负担等二维显微镜技术。 总体而言，EDFM和HSI提供若干优点优于常规纳米成像和表征技术，如TEM，HAADF和DIC。 EDFM / HSI允许更快的图像采集和分析，从而节省了时间和成本相比，更密集4.便利tional技术。此外，对于EDFM / HSI样品制备通常都极小和非破坏性，从而节省时间，还允许对于给定的样品的更灵活的分析，因为它随后可以用于其它技术。此外，恒指多才多艺，允许许多组成的纳米材料的分析，并在各种基质。该研究团队正在努力改进此描述的其他材料和样品类型，包括深入评估该技术的特异性的方法。由研究小组正在调查的一个关键下一步是对传统的黄金标准，这些技术验证（ 例如 ，拉曼光谱，TEM，SEM）的材料和感兴趣的组织类型。

作为纳米材料在各种行业和应用越来越多地使用，有需要一种更快速，经济实惠，并方便比传统方式，诸如电子显微镜的纳米成像和表征方法。为了显现与细胞，组织，和生命系统，许多光学技术已经被采用，包括微分干涉对比（DIC）显微1和渐逝场为基础的方法，例如全内反射（TIR）或近纳米颗粒（NP）的相互作用场扫描光学显微镜（NSOM）2,3。然而，这些高端的分析方法，掉大部分非专业实验室4拿不到的地方。电子显微镜，包括透射电子显微镜（TEM），也被用来研究的NP与细胞5,6,7,8-相互作用。高角环形暗场（HAADF）扫描透射电子显微镜已经被用于研究纳米粒子的相互作用与病毒9。共聚焦显微镜是用于研究NP-细胞相互作用10另一种流行的技术。 近年来，暗场为基础的超光谱成像（HSI）技术已经被用作用于在生物基质11就读纳米粒一种很有前途的分析工具。 HSI系统产生空间和光谱数据，被称为一个超立方体或datacube 12的三维表示。光谱变化的空间映射用于材料识别。可以产生和对比未知样品用作参考库已知材料光谱图。一与HSI系统的主要优点是它能够成像光谱结合，由此建立的位置和未知的NP的分布在体内或离体以及它们链接到已知的，类似的组合物的另一个参考颗粒。 有几个优点采用HSI系统比传统成像技术:最小的样品制备是必需的;样品制备是通常的非破坏性的性质;图像采集和分析是快;该技术具有成本效益的13;和空间分布的混合组合物和/或复杂基质中的化合物的分析更容易完成14。 纳米材料的研究，涉及珍贵样品，其中最​​重要的考虑因素之一是一种非破坏性成像方法，其允许所述潜在重复检查由一种或多种方法的样品的可用性。重复或多个分析可期望开发综合数据集，不会从一个单一的方法是可用的。为了对此，研究其光学特性是分析样品的最安全的方式。通过使用增强暗视野显微镜（EDFM）和HSI系统，研究样品的光学响应 - 即reflectance，而且吸光度和透射率-特征鉴定和表征可以执行15。潜在的表征端点包括相对尺寸和样品中的纳米颗粒或附聚物和纳米颗粒的分布的形状的评估。 在本文中，我们描述了映射方法专门用于金属氧化物纳米颗粒在死后组织使用基于被称为光谱角映射器（SAM）的像素光谱匹配算法一个HSI系统。我们选择这种特定应用，因为它具有以补充当前和未来的体内纳米毒理学研究，其中的动物模型被用来评估曝光对健康的影响，以工程纳米材料的潜能。这种方法的应用也可以通知纳米药物递送研究了利用组织或动物模型。尤其是，纳米颗粒的吸收，分布，代谢和排泄整个örgans和组织可能与此系统进行调查。各种各样的应用正在在生物医学研究11调查使用。 该方法可以被用于不同的生物样品（如各种组织的类型，支气管肺泡灌洗样本，和血涂片），该已暴露于纳米颗粒的各种元素组成16-19的评估。此外，这种方法是用于研究纳米颗粒的生物分布在体内和体外 ，这是相关的纳米级药物递送研究11是有用的。以外的生物样品，EDFM和HSI可以用来评估纳米颗粒环境样品，如废水20个在。职业暴露评估可通过使用此技术来促进，以及，因为它可被用来评估在防止纳米颗粒穿透的个人防护设备的功效。此外，研究忒凌晨目前正在开发一个类似的EDFM和HSI协议的纳米颗粒的职业暴露评估收集的过滤介质样品的评价。而准备EDFM和HSI这些不同类型的样品可能会有所不同，但重要的是它们的制备，因为它们可以由光学系统很容易地可视化的方式。典型地，样品应制备为如果它会经由传统明场显微可视化。有市售的11几个高光谱成像系统。

<table><tbody><tr><td>CytoViva 150 Unit (condenser)</td><td>CytoViva (Auburn, AL)</td><td></td><td>mounted to Olympus BX43 microscope</td></tr><tr><td>Olympus BX43 Microscope - Analyzer Slot - HSI with 10x and 40x air objectives and 100X oil immersion objective</td><td>obtained through CytoViva (Auburn, AL)</td><td></td><td>for use with CytoViva 150 Unit condenser</td></tr><tr><td>Dagexcel-M Digital Firewire Camera - Cooled; includes Exponent 7 software</td><td>obtained through CytoViva (Auburn, AL)</td><td></td><td>enhanced darkfield camera and software</td></tr><tr><td>CytoViva Hyperspectral Imaging System 1.4; includes Pixelfly hyperspectral camera, XY stage controller, ENVI hyperspectral imaging software</td><td>obtained through CytoViva (Auburn, AL)</td><td></td><td>hyperspectral camera and software</td></tr><tr><td>cleanroom cleaned glass microscope slides (glass B slides)</td><td>Schott NEXTERION</td><td>1025087</td><td>reduced debris and artifacts compared to conventional glass microscope slides for optimal imaging</td></tr><tr><td>cleanroom cleaned glass microscope coverslips (#1.0; 22 mm x 22 mm x&amp;nbsp; 1.45 mm)</td><td>Schott NEXTERION</td><td>custom</td><td>reduced debris and artifacts compared to conventional glass coverslips for optimal imaging</td></tr><tr><td>type A microscopy immersion oil</td><td>Fisher Scientific</td><td>12368B</td><td>multiple suppliers</td></tr><tr><td>70% isopropanol in water</td><td></td><td></td><td>multiple suppliers</td></tr><tr><td>ImageJ software</td><td>National Institutes of Health (NIH)</td><td></td><td>free open-source software online download</td></tr><tr><td>metal oxide nanoparticles</td><td>supplied to the research team by industrial partners</td><td></td><td>alumina, silica, and ceria nanoparticles in aqueous suspensions. Due to a Non-Disclosure Agreement between the authors and industry partners, further product information cannot be disclosed.</td></tr></tbody></table>

identification of metal oxide nanoparticles in histological samples by enhanced darkfield microscopy and hyperspectral mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

高光谱显微镜是它能够识别材料类似于分光方式的能力非常有用。 正如图1中，每一种材料具有几个特性光谱和其反射率的整体形状，其是唯一的。此外， 图1示出的光谱轮廓的基质依赖性性质:用于每个组织学组织样品中的三个金属氧化物（上图）的光谱范围是从每个在含水悬浮液中的三个金属氧化物的光谱曲线（不同底部面板）。通过在同一矩阵映射特征光谱型材未知样品，该技术是用于确定存在的材料或不存在下是有用的，并且还可以半定量样品中比较材料的相对量。 从阳性对照皮肤样本中创建的参考谱库适合映射实验皮肤样本。 正如图2中，参考光谱库很好地映射到相应的阳性对照和不映射到相应的阴性对照。 该技术的最显著优点是其以检测感兴趣的材料低的水平在样品中，以及从污染物区分他们的能力。例如，单独的纳米管可在肺吸入剂量低至40微克在20-30克鼠25的过程中观察到图3演示了这种组织学皮肤样品中:而一些颗粒是暗场光学图像中容易地明显（列2），其它的是使用高光谱成像（第3列），可能已使用亮视野显微镜（列1）或其它方法检测到的遗漏。采用暗场恒指也有助于提高特异性高于简单的明显微镜。然后这些图像可以受到分析更多量化形式，正通过ImageJ的otably颗粒分析。 增强暗视野显微镜具有若干不同的应用，即使在没有高光谱成像。第一个是它能够产生高品质，高对比度图像的能力。这最好由图3证实，其中感兴趣粒子相比，其明对应易于识别。这些图像也可以是受数量颗粒分析，尽管在不存在高光谱映射，需要谨慎，以避免来自感兴趣的颗粒混淆的污染物颗粒。 <img alt="图1" src="/files/ftp_upload/53317/53317fig1.jpg" /> 相比于谱库（悬浮纳米颗粒）图1.参考谱库（组织基质）。这个数字表明，从产生楠参考谱库的重要性在同一矩阵作为实验样品的兴趣omaterials。第一行显示的参考谱库（RSL）在这项研究（氧化铝，二氧化硅和氧化铈），每一种材料。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53317/53317fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 此RSL是由在这个协议中，其中阳性对照组织样品进行筛选的阴性对照组织样品中描述的方法来创建。第二行显示了由在载玻片上在液体悬浮感兴趣的纳米颗粒在光谱的库（SL）。为氧化铝，在500和650的波长的双峰峰被发现在RSL，而更弯曲的，并且较少明显峰的混合物在周围600为二氧化硅，峰值出现在氧化铝的NP悬浮SL在大约650一个波长为本RSL中的，而更弯曲的，并且较少明显峰的混合物在腌肉见于硅石NP悬浮SL次600。氧化铈，双峰峰被发现的RSL中的520和620的波长，而较少明显峰的混合物在周围560这表明，在该纳米颗粒包埋于基质创建看出，在氧化铈的NP​​悬浮SL在光谱的移位选择的控件时创建对SL为具体实验，可以是相当大的。 <img alt="图2" src="/files/ftp_upload/53317/53317fig2.jpg" /> 图2.参考光谱库（组织基质）被映射在阳性对照和阴性对照猪皮肤组织 。该图中作为确认的RSL适当的用于映射到实验样品，在每个RSL（左列）以及映射到其相应的阳性对照（中间列），不映射到其相应的阴性对照（右列）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53317/53317fig2large.jpg" target="_blank">请点击ħ</a>ERE查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53317/53317fig3.jpg" /> 图3.高光谱测绘实验猪的皮肤组织暴露于金属氧化物纳米颗粒 。行上下对应于不同层的皮肤，从浅到最深层:表皮，真皮​​和皮下组织，分别。第一行示出了从皮肤样品被暴露于在悬浮液中的氧化铝纳米颗粒的表皮的角质层 ;第二行是矽纳米颗粒悬浮液;和第三行与铈土的纳米颗粒在悬浮液中。每列描绘成像的不同技术皮肤的相同区域。第一列对应于明显微镜（40X MAG;比例尺= 50微米），在封闭在一个红色的方形区域被放大（100X MAG;比例尺= 10μm），以增强暗视野显微镜（EDFM），在第二列中，其中，白箭头指向高对比度的NP。用超光谱照相机（HSI），获得第三列，示出了相同的高对比度的NP（白色箭头）。 EDFM和HSI影像拍摄放大​​100倍;比例尺= 10微米。第四列显示映射对各自的RSL每次曝光组，其中氧化铝纳米颗粒都显示为绿色，二氧化硅纳米颗粒在红色，氧化铈纳米颗粒的蓝筹股形象黄色，分别。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53317/53317fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping at JoVE.com

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

identification-metal-oxide-nanoparticles-histological-samples

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

12.4K 次观看.  SUNY Polytechnic Institute, Colleges of Nanoscale Science and Engineering. Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

视频: Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1&#160;to 24 hr&#160;after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 &#956;m. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response:&#160;elemental, molecular, or cellular.

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 &#956;m and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

激光诱导击穿光谱：对于纳米粒子的映射和定量的器官组织的新方法

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) ...

科研

工程学

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

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

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

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

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

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

化学

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

单金属纳米粒子的高分辨率物理特性

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

生物化学

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy.

基于传输的野武学型差分干涉对比显微镜对等离子体纳米粒子的光谱处理

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using ...

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 ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

生物工程

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar energy and fuel generation, and electrochemical energy storage. Nanoscale characterization of such interfaces has recently been achieved using cryogenic electron microscopy, thereby providing a new path to advancing our fundamental understanding of interface processes.
This contribution provides a practical guide to mapping the structure and chemistry of solid-liquid interfaces in materials and devices using an integrated cryogenic electron microscopy approach. In this approach, we pair cryogenic sample preparation which allows stabilization of solid-liquid interfaces with cryogenic focused ion beam (cryo-FIB) milling to create cross-sections through these complex buried structures. Cryogenic scanning electron microscopy (cryo-SEM) techniques performed in a dual-beam FIB/SEM enable direct imaging as well as chemical mapping at the nanoscale. We discuss practical challenges, strategies to overcome them, as well as protocols for obtaining optimal results. While we focus in our discussion on interfaces in energy storage devices, the methods outlined are broadly applicable to a range of fields where solid-liquid interface play a key role.

Cryogenic Focused Ion Beam (FIB) and Scanning Electron Microscopy (SEM) techniques can provide key insights into the chemistry and morphology of intact solid-liquid interfaces. Methods for preparing high quality Energy Dispersive X-ray (EDX) spectroscopic maps of such interfaces are detailed, with a focus on energy storage devices.

通过冷冻聚焦离子束铣削与扫描电子显微镜和光谱学耦合，对液固界面进行纳米级表征

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar ...

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

使用多模态非线性光学显微镜对纳米颗粒的细胞摄取进行生物分子成像

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光谱遥感制图

摘要

探索更多视频

此视频中的章节

激光诱导击穿光谱：对于纳米粒子的映射和定量的器官组织的新方法

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

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

单金属纳米粒子的高分辨率物理特性

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

基于传输的野武学型差分干涉对比显微镜对等离子体纳米粒子的光谱处理

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

相关轻型和电子显微镜使用量子点纳米粒子

通过冷冻聚焦离子束铣削与扫描电子显微镜和光谱学耦合，对液固界面进行纳米级表征

使用多模态非线性光学显微镜对纳米颗粒的细胞摄取进行生物分子成像

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

摘要

探索更多视频

此视频中的章节

激光诱导击穿光谱：对于纳米粒子的映射和定量的器官组织的新方法

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

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

单金属纳米粒子的高分辨率物理特性

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

基于传输的野武学型差分干涉对比显微镜对等离子体纳米粒子的光谱处理

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

相关轻型和电子显微镜使用量子点纳米粒子

通过冷冻聚焦离子束铣削与扫描电子显微镜和光谱学耦合，对液固界面进行纳米级表征

使用多模态非线性光学显微镜对纳米颗粒的细胞摄取进行生物分子成像

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光谱遥感制图