Name: 视频: Video-rate Scanning Confocal Microscopy and Microendoscopy
Uploaded: 2011-10-20T13:00:00.000+00:00
Duration: 14 min 10 s
Description: 27.7K 次观看. Harvard University. 定制的，实时的共焦扫描成像系统的完整的建筑描述。该系统，该系统可以很容易地用率视频显微镜和内窥镜，使成像的几何形状和应用程序使用标准的商用共焦系统成本的一小部分，而不是访问数组。

作者想感谢他们支持这一项目ThorLabs。 AJN希望承认的美国国家科学基金会研究生奖学金的支持。 这项工作是部分资金由国立卫生研究院通过美国国立卫生研究院主任的新的创新奖项目，授予1 DP2 OD007096 - 01。的新的创新奖计划的<a href="http://nihroadmap.nih.gov/newinnovator/">信息</a> http://nihroadmap.nih.gov/newinnovator/ 。作者想感谢汤姆海斯使用哈佛电子实验室。

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	<li>Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of biological confocal microscopy. , 985-985 (2006).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nature Reviews Molecular Cell Biology. 2, 444-456 (2001).</li><li>Klonis, N., Rug, M., Harper, I., Wickham, M., Cowman, A., Tilley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+photobleaching+analysis+for+the+study+of+cellular+dynamics.">Fluorescence photobleaching analysis for the study of cellular dynamics.</a> European Biophysics Journal. 31, 36-51 (2002).</li><li>Stephens, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Microscopy+Techniques+for+Live+Cell+Imaging.">Light Microscopy Techniques for Live Cell Imaging.</a> Science. 300, 82-86 (2003).</li><li>McMahon, A., Supatto, W., Fraser, S. E., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Analyses+of+Drosophila+Gastrulation+Provide+Insights+into+Collective+Cell+Migration.">Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration.</a> Science. 322, 1546-1550 (2008).</li><li>Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled+controls+cell+polarity+during+Xenopus+gastrulation.">Dishevelled controls cell polarity during Xenopus gastrulation.</a> Nature. 405, 81-85 (2000).</li><li>Laemmel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibered+Confocal+Fluorescence+Microscopy+(Cell-viZio)+Facilitates+Extended+Imaging+in+the+Field+of+Microcirculation.">Fibered Confocal Fluorescence Microscopy (Cell-viZio) Facilitates Extended Imaging in the Field of Microcirculation.</a> Journal of Vascular Research. 41, 400-411 (2004).</li><li>Moussata, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+confocal+laser+endomicroscopy.">The confocal laser endomicroscopy.</a> Acta Endoscopica. 39, 448-451 (2010).</li><li>Dunbar, K., Canto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+endomicroscopy.">Confocal endomicroscopy.</a> Current Opinion in Gastroenterology. 24, 631-637 (2008).</li><li>Udovich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+background+and+transmission+characteristics+of+fiber+optic+imaging+bundles.">Spectral background and transmission characteristics of fiber optic imaging bundles.</a> Applied optics. 47, 4560-4568 (2008).</li></ol>

Thorlabs公司赞助制作这个视频，这个视频

此视频率成像系统，使约8 kHz的一个共振的振镜镜操作使用。谐振镜可以很响亮，在满功率运行时，他们的高音可以在足够的曝光时间的麻烦甚至是危险的。虽然没有表现出在这里，它建议在一个透明的情况下屏蔽谐振振镜镜显著减少系统体积和/或穿适当的听力保护装备，如耳塞，。 共振的振镜镜扫描正弦模式。然而，采集卡卡读取信号在水平和垂直两个方向假设一个完全线性扫描率。由于正弦扫描减慢扫描边缘，图像压缩失真可以观察到沿快速图像轴（水平）。来减少这种问题的方法之一是故意显著驱动器的谐振振镜镜扫描范围都比较大继电器镜头直径。在这一过程中，只有近线性的正弦扫描模式的中央扫穿过样品，尽量减少图像失真。另一种方法是将后处理收集到的图像线性快轴。这可以由成像已知的荧光模式（如电网），并使用已知的模式尺寸创建一个unwarps收集到的图像处理脚本。 这种特殊的扫描系统的目的是为在体内成像的目的，往往需要一个堂堂正正的面向率视频显微镜。细胞成像实验，倒置显微镜是比较典型的使用。这里介绍的设计可以很容易地更改为建立这样一个倒置显微镜，所有需要的是最后2"直径镜的旋转。定向镜子向下直接扫描光束，而是镜子可以直接向上的光束。配售的客观镜头吨镜与样品阶段相同的距离，将允许在一个倒置的几何结构成像。如果成像系统正在修建显微成像单纯，没有任何理由"折"显微镜的设计，在所有垂直。相反，整个扫描系统，可以建在一个单一物镜方向平行光表的水平面包板。 请注意，在这个构建的显微镜使用一个固定的针孔配置;，而这提供了最大的构建简单和便于对齐，渴望一个更灵活的系统的用户可能会考虑把一个变量的针孔，可以发现大多数商业共聚焦显微镜。通过允许用户调整大小的针孔，以弥补不同排放强度的样品，这使用户能够更好地优化一个给定的样本的信号强度和分辨率之间的权衡。 这架CHOICE显微镜选定的图像纤维是重要的。我们建议使用住友连贯的形象由于他们密切的光纤芯间距和相对低的自体荧光纤维。图片纤维生产由藤仓已发现有10的自体荧光，这可以从样品压倒弱荧光信号和限制microendoscope最终灵敏度高的金额。住友生产的纤维，如在这个特别的设置使用8 - 30N，比藤仓等值的自体荧光的水平要低得多。虽然传送次数纤维束可能被视为对内窥镜的吸引力，其设计通常地方个别纤芯相距甚远，这意味着，纤芯稀疏的样本对象，离开了显著地区的潜在利益。 最后，应该指出，虽然在显微镜这里将各种有用在体外和体内 applicati组件，可用于创建一个全功能的商业系统的成本的一小部分，它没有功能，如透射光检测，取景目镜，或一个非共焦广角epifluorescence光束路径。虽然有可能构建一个系统从头这些功能，读者希望这样的系统可能会希望修改现有的商业系统，以满足他们的需求，而不是启动一个全新的构建。

<table border="1"><tbody><tr><td> 部分名称 </td><td> 制造商 </td><td> 编号 </td><td> 规格 </td><td> 数量 </td></tr><tr><td> 515 nm带通滤波器</td><td>浓度</td><td> HQ515/50M </td><td> 46的FWHM </td><td> 1 </td></tr><tr><td> 25.4毫米的消色差双峰透镜直径。 x 50毫米佛罗里达州，氟化镁涂层</td><td>爱特蒙特光学</td><td> NT49 - 766 </td><td></td><td> 1 </td></tr><tr><td> 25.4毫米的消色差双峰透镜直径。 x76.2毫米FL，氟化镁涂层</td><td>爱特蒙特光学</td><td> NT49 - 768 </td><td></td><td> 1 </td></tr><tr><td> 25.4毫米的消色差双峰透镜直径。 x88.9毫米FL，氟化镁涂层</td><td>爱特蒙特光学</td><td> NT49 - 769 </td><td></td><td> 2 </td></tr><tr><td> 50毫米的消色差双峰透镜直径。 x 300毫米佛罗里达州，氟化镁涂层</td><td>爱特蒙特光学</td><td> NT45 - 179 </tD&gt; <td></td><td> 1 </td></tr><tr><td> 8 kHz的R高频率的光学扫描仪</td><td>光电产品公司（EOPC） </td><td> SC - 30 </td><td> 8千赫</td><td> 1 </td></tr><tr><td> AGC的驱动程序</td><td>光电产品公司（EOPC） </td><td> ACG:8K </td><td></td><td></td></tr><tr><td> H7422 - PA的感光模块</td><td>滨松</td><td> H7422 - PA </td><td>电流限制建议</td><td> 1 </td></tr><tr><td> M9012电源供应</td><td>滨松</td><td> M9012 </td><td> H7422 - PA </td><td> 1 </td></tr><tr><td>慧聪的PL APO政务司司长目的</td><td>徕卡</td><td> 11506284 </td><td> 10x/0.40 </td><td> 1 </td></tr><tr><td> Solios EA / XA采集卡卡</td><td>的Matrox </td><td> Solios EA / XA </td><td> MIL所需软件;代工互连建议</td><td> 1 </td></tr><tr><td> 12V电源</td><tD&gt;明纬</td><td> LPV - 100 - 12 </td><td> +12 V，8.5A </td><td> 1 </td></tr><tr><td> 5倍显微物镜</td><td>新港</td><td> M - 5X </td><td> 0.10不适用，25.4毫米的焦距</td><td> 1 </td></tr><tr><td>相干图像纤维</td><td>住友商事</td><td> 8 - 30N </td><td></td><td> 1 </td></tr><tr><td> 1 / 4"-20帽螺钉和硬件套件</td><td> ThorLabs </td><td> HW - KIT2 </td><td></td><td> 1 </td></tr><tr><td> 100微米安装针孔</td><td> ThorLabs </td><td> P100S </td><td>建设空间滤波器的理想选择</td><td> 1 </td></tr><tr><td> 30毫米笼立方钳</td><td> ThorLabs </td><td> B6C </td><td></td><td> 1 </td></tr><tr><td> 30毫米笼系统立方，4路</td><td> ThorLabs </td><td> C4W </td><td></td><td> 1 </td></tr><tr><td> 406纳米，5毫瓦，B PIN码，单模光纤尾纤的激光二极管，FC / PC </td><td> ThorLabs </td><td>大号PS - 406 - FC </td><td>产品过时，取而代之的LP405 - SF10 </td><td> 1 </td></tr><tr><td> 5分钟环氧，1盎司</td><td> ThorLabs </td><td> G14250 </td><td></td><td> 1 </td></tr><tr><td> 6轴运动光山</td><td> ThorLabs </td><td> K6X </td><td></td><td> 1 </td></tr><tr><td> 8-32帽螺钉和硬件套件</td><td> ThorLabs </td><td> HW - KIT1 </td><td></td><td> 1 </td></tr><tr><td> 8-32紧固螺栓和硬件套件</td><td> ThorLabs </td><td> HW - KIT3 </td><td></td><td> 1 </td></tr><tr><td>适配器与外部RMS线程和内部的SM1线程</td><td> ThorLabs </td><td> SM1A4 </td><td></td><td> 1 </td></tr><tr><td>调整。 FC / PC和FC / APC准直仪，F = 2.0毫米，ARC:400-600纳米</td><td> ThorLabs </td><td> CFC - 2X - </td><td> F = 2.0毫米</td><td> 1 </td></tr><tr><td>可调光纤准直器适配器，SM1一起螺纹</td><td> ThorLabs </td><td> AD9.5F </td><td></td><td> 1 </td></tr><tr><td>铝面包板，12"× 18"× 1 / 2" </td><td> ThorLabs </td><td> MB1218 </td><td> 1 / 4"-20螺纹</td><td> 2 </td></tr><tr><td>台式激光二极管/ TEC控制器</td><td> ThorLabs </td><td> ITC4001 </td><td> 1 A/96 W </td><td> 1 </td></tr><tr><td> DMLP 425纳米长通分色镜</td><td> ThorLabs </td><td> DMLP425 </td><td></td><td> 1 </td></tr><tr><td>运动摩O1"光学</td><td> ThorLabs </td><td> KM100 </td><td></td><td> 3 </td></tr><tr><td> ThorLabs光纤尾纤激光二极管LD / TEC安装</td><td> ThorLabs </td><td> LM9LP </td><td></td><td> 1 </td></tr><tr><td> O18毫米光学镜头山</td><td> ThorLabs </td><td> LMR18 </td><td>包括其中一个扣环</td><td> 1 </td></tr><tr><td> 2"光学镜头底座</td><td> ThorLabs </td><td> LMR2S </td><td>随着内部和外部的线程;扣环包括</td><td> 2 </td></tr><tr><td>迷你系列笼大会棒，6"长，O4毫米，数量1。 </td><td> ThorLabs </td><td> SR6 </td><td></td><td> 4 </td></tr><tr><td> Ø1.0"基座支柱邮政8-32水龙头，1个"长</td><td> ThorLabs </td><td> RS1P8E </td><td></td><td> 4 </td></tr><tr><td> O1"支柱邮政支线，长度= 0.5 </td><td> ThorLabs </td><td> RS05 </td><td></td><td> 4 </td></tr><tr><td> O1"支柱邮政扩展，长度= 0.75" </td><td> ThorLabs </td><td> RS075 </td><td></td><td> 4 </td></tr><tr><td> O1"保护银镜，厚为3.2毫米</td><td> ThorLabs </td><td> ME1 - P01 </td><td></td><td> 1 </td></tr><tr><td> O1"SM1一起旋转可调对焦元素，L = 1" </td><td> ThorLabs </td><td> SM1V10 </tD&gt; <td></td><td> 1 </td></tr><tr><td> O2"保护银镜，厚为3.2毫米</td><td> ThorLabs </td><td> ME2 - P01 </td><td></td><td> 2 </td></tr><tr><td> P100S - Ø100微米装针孔</td><td> ThorLabs </td><td> P100S </td><td></td><td> 1 </td></tr><tr><td>宝来低漂移O1"运动镜山</td><td> ThorLabs </td><td> POLARIS - K1 </td><td>低漂移</td><td> 1 </td></tr><tr><td> SM1镜头管，L = 1" </td><td> ThorLabs </td><td> SM1L - 10 </td><td>包括其中一个扣环</td><td> 4 </td></tr><tr><td> SM1螺纹30毫米笼板，0.35"厚</td><td> ThorLabs </td><td> CP02 </td><td></td><td> 2 </td></tr><tr><td> SM1一起M25光学组件的线程适配器</td><td> ThorLabs </td><td> SM1A24 </td><td>外部SM1主题和内部M25.5x0.5主题</td><td> 1 </td></tr><tr><td>小光束直径ETER振镜系统</td><td> ThorLabs </td><td> GVSM001 </td><td></td><td> 1 </td></tr><tr><td>小夹紧叉</td><td> ThorLabs </td><td> CF125 </td><td> 1 / 25"沉头槽，普遍</td><td> 15 </td></tr><tr><td>空间过滤系统</td><td> ThorLabs </td><td> KT310 </td><td>针孔单独出售</td><td> 1 </td></tr><tr><td> TE冷却的坐骑为5.6和9毫米的激光</td><td> ThorLabs </td><td> TCLDM9 </td><td></td><td> 1 </td></tr><tr><td>为面包板的垂直支架</td><td> ThorLabs </td><td> VB01 </td><td>每个</td><td> 2 </td></tr><tr><td>计划的复消色差透镜</td><td>蔡司</td><td> 1101-957 </td><td> 20x/0.75 NA </td><td> 1 </td></tr></tbody></table>

video-rate scanning confocal microscopy and microendoscopy

共聚焦显微镜在生物学和生物医学科学已成为宝贵的工具，实现快速，高灵敏度和高分辨率复杂系统的光学切片。共聚焦显微镜是经常使用的，例如，研究在活细胞2-4的特定细胞的目标 1，监控动态，5,6整个生物体的三维可视化的三个演变。共聚焦成像系统，如焦microendoscopes，扩展，使高分辨率成像在体内 7，目前正在应用到疾病的成像和设置在 8,9临床诊断。 通过创建所谓的"光学"，利用简单的几何光学共聚焦显微镜提供立体的决议。在一个标准的宽视场显微镜，从样品产生的荧光是由物镜收集，并直接传达到一个探测器。虽然接受能够对薄样品成像变得模糊，厚样品上方和下方的客观焦平面产生荧光。相比之下，共聚焦显微镜使虚拟光学切片，样品，拒绝了焦光建立样品的高分辨率三维交涉。 共聚焦显微镜检测光束路径中使用共焦光圈实现这一壮举。从样品收集客观的荧光是通过扫描镜，并通过主分色镜，镜子精心挑选的反映，如激光激发束的波长较短，同时传递的时间越长，斯托克斯位移的荧光发射中继。这长波长的荧光信号，然后通过一个针孔是在一个平面上的位置正好与物镜的焦平面共轭两侧的一对镜片。从对象的焦点卷收集的光子是平行由物镜和集中通过针孔共焦镜头。高于或低于焦 ​​平面产生荧光，因此将不平行正确，并不会通过共焦针孔1，创建一个光学部分，只能从显微镜对焦可见。 （ 图1）。因此，针孔有效地作为一个在焦平面的虚拟光圈，检测排放限制只有一个有限的空间位置。 现代商业的共聚焦显微镜为用户提供了完全自动化操作，使以前复杂的成像过程相对简单和易于获得的。尽管这些系统的灵活性和力量，商业共聚焦显微镜是不能很好地适用于所有共聚焦成像的任务， 如在许多体内成像应用， 。没有能够创建定制的成像系统，以满足他们的需求，重要的实验，可以留出反应h到许多科学家。 在这篇文章中，我们提供了一种习俗，视频率共聚焦成像系统的基本组成部分的完整的施工一步一步的方法。直立的显微镜，将兴建一个共振的振镜镜提供快速扫描轴，而一个标准速度的谐振振镜镜扫描慢轴。要创建一个精确的扫描光束在物镜焦点，这些反射镜将定位在所谓的远心飞机使用四个继电器镜片。共聚焦显微镜检测将使用一个标准，现成的，现成的光电倍增管（PMT）完成，并使用Matrox卡采集卡和附带的软件将被捕获并显示图像。

Watch this Scientific Journal Video about Video-rate Scanning Confocal Microscopy and Microendoscopy at JoVE.com

Video-rate Scanning Confocal Microscopy and Microendoscopy

定制的，实时的共焦扫描成像系统的完整的建筑描述。该系统，该系统可以很容易地用率视频显微镜和内窥镜，使成像的几何形状和应用程序使用标准的商用共焦系统成本的一小部分，而不是访问数组。

视频率扫描共聚焦显微镜和内窥镜

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

video-rate-scanning-confocal-microscopy-and-microendoscopy

Research

JoVE Journal

Bioengineering

27.7K 次观看.  Harvard University. 定制的，实时的共焦扫描成像系统的完整的建筑描述。该系统，该系统可以很容易地用率视频显微镜和内窥镜，使成像的几何形状和应用程序使用标准的商用共焦系统成本的一小部分，而不是访问数组。

视频: Video-rate Scanning Confocal Microscopy and Microendoscopy

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

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

刀口扫描显微镜的样品制备，图像和分析协议

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture cellular behaviors involved in developmental processes.&#160; The ability to track and follow cell movements is limited by sample movements that occur due to drift of the sample or, in some cases, growth during image acquisition. Tracking cells in datasets affected by drift and/or growth will incorporate these movements into any analysis of cell position. This may result in the apparent movement of static structures within the sample. Therefore prior to cell tracking, any sample drift should be corrected. Using the open source Fiji distribution 1 &#160;of ImageJ 2,3 and the incorporated LOCI tools 4, we developed the Correct 3D drift plug-in to remove erroneous sample movement in confocal datasets. This protocol effectively compensates for sample translation or alterations in focal position by utilizing phase correlation to register each time-point of a four-dimensional confocal datasets while maintaining the ability to visualize and measure cell movements over extended time-lapse experiments.

Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time.

样品漂移校正继4D共焦时间推移成像

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

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 ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

在中短肛解剖，显微扫描和分析形态

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

用 Widefield 高含量分析系统实现荧光标记组织的自动滑动扫描和分割

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...