该项目是由美国国立卫生研究院/ NINDS（＃1R01 - NS54252），NSF（＃0905041，＃0079874），得克萨斯州的先进技术计划（ATP - 000512 - 0146 - 2001，＃ATP - 000512 - 0261 - 2001），得克萨斯州A和中号研究基金会，大学计算机科学与工程在得克萨斯A＆M大学和3Scan系。我们想感谢陈智思梅萨（科技之星技术）KESM仪器的技术咨询和支持。的设计和实施KESM的主要部分是由Bruce H.麦考密克，在2007年去世。

1。试样准备：高尔基 - 考克斯<ol><li>紧随该协议的协议。Mayerich等7。 </li><li>的C57BL/6J小鼠深深麻醉用异氟醚吸入麻醉，然后断头。 </li><li>大脑中删除，并放入一个高尔基考克斯固定的解决方案（1％铬酸钾，1％重铬酸钾，去离子水和1％的升汞）。 </li><li>大脑是留在黑暗中的高尔基体考克斯解决方案，在室温，10至16周。 </li><li>大脑在黑暗中过夜去离子水冲洗。 </li><li>沉浸在铵去离子水在室温下，在黑暗中为7至10天5％的氢氧化钠溶液冲洗大脑。这个漫长的准备时间，以确保整个大脑的渗透，使黑色沉淀物有机会在组织完全形成。 </li><li>大脑是在去离子水冲洗干净，再在室温为4小时和T母鸡通过乙基醇梯度脱水：50％和70％的冰箱（4℃），和85％，95％（3变化），和100％（4至5变化）在室温下。大脑是留在每24小时的解决方案。 </li><li>然后把脱水的大脑是在丙酮（3至4的变化，每一天），牢牢丙酮比为1:2，1:1，2:1，终于在丙酮100％的牢（3变化，每次通宵），在冰箱中，（4℃ ）。 </li><li>最后，处理后的大脑是嵌入在100％牢，并加热至60 ° C，连续3天（参见2雅培和Sotelo嵌入协议）。 如果标本在LR白嵌入式然后通过三个转变LR白色的解决方案，均在冰箱过夜，这是一个标准程序聚合前的标本是从过去的100％的乙醇溶液转移。完成三个转变，以确保充分浸润。标本是T母鸡转移到新鲜LR的白色聚合在一个密闭容器中，在60 ° C为24小时，这是正确的聚合所需的时间和温度。 </li><li>图图1显示了高尔基体考克斯染色脑嵌入在环氧树脂。 </li><li>固化后的试样块，然后安装在金属试样用环氧环，块的两侧，作为必要的修剪（见图2）。 </li></ol> 2。试样制备：尼斯尔<ol><li>鼠标是深深使用氯胺酮和甲苯噻嗪（1.7毫克氯胺酮/甲苯​​噻嗪0.26毫克每20克体重）腹腔注射麻醉，然后用50毫升室温灌注transcardially磷酸盐缓冲液（pH值7.4），250毫升的空间温度10％的中性福尔马林缓冲液（pH值7.4）。最后用3.0毫升未经稀释的印度墨水。 </li><li>全身灌注生理盐水和固定液是必要的，以清除血液和心血管系统的修复的TI<html> ssues。印度墨汁灌注是必要的，完全填补了心血管系统的血管。 </li><li>通过一系列分级乙基醇（25％-100％）造成的大脑，然后脱水，然后在牢塑料嵌入在1.8-1.9以上的协议。如果LR白了就是包埋剂，然后在1.10以上的协议是其次。 </li></ol> 4。试样制备：通用品种，一般机关<ol><li>一般情况下，可以用固定为10％中性缓冲福尔马林或4％多聚甲醛。 </li><li>对于小型组织卷，而不是灌注扩散，建议固定和染色。在小生物体或器官的情况下，染色也可以做到在脱水过程中。 </li><li>嵌入协议是与上面相同，解决方案渗透的时代，虽然可以减少器官或生物体，比整个鼠脑小。 </li></ol>jove_title“&gt; 5。KESM设置和成像<ol><li>关闭泵，打开舞台上，打开相机，打开照明灯。 </li><li>提高刀和客观。 </li><li>安装试样环。 </li><li>测量试样的尺寸和KESM阶段Controller2应用程序创建一个配置文件。 </li><li>开始KESM阶段Controller2和初始化阶段。 </li><li>下刀/目标，打开泵。 </li><li>焦点目标和摄像头，眼镜直通车转动调焦旋钮，调整摄像机的视野相对刀刃。 </li><li>按[返回]发起成像。 </li><li>参见图。 3-8。见9的技术细节，7。 </li></ol> 6。图像处理和数据准备<ol><li> KESM栈处理器可执行文件复制到数据文件夹下的配置文件的文件夹（00000，00001等）。 </li><li> KESM栈处理器运行删除照明神器和正常化的背景强度图像。重复子文件夹中的所有数据。 </li><li>准备从数据多尺度瓷砖设置和KESM脑Atlas的Web服务器中的文件上传，并成立。 </li><li>见图。 9。 </li></ol> 7。数据可视化和分析<ol><li>使用KESM脑图谱的可视化。转到<a href="http://kesm.cs.tamu.edu">http://kesm.cs.tamu.edu</a> ，并选择适当的地图集。 <ol><li>在谷歌地图导航。 </li><li>在谷歌地图的放大和缩小。 </li><li>要进入到一个不同的深度，选择从下拉菜单中的每一步有多深，然后点击或者[+]或[ - ]增加或减少Z深度。 </li><li>通过使用下拉菜单调整叠加数量。 </li><li>通过使用下拉菜单调整覆盖的时间间隔。 </li><li>见图。 11。 </li></ol></li><li>可视化使用MeVisLab。 <ol><li>启动MeVisLab。 </li><li>创建一个新项目。 </li><li>在下面的，新的模块，可以创建由Ty平安在命令中的模块名称。 </li><li>创建模块[Compose3Dfrom2DFiles]，并转到所需的文件夹。输入文件中的字符串，然后单击[GetFileList]。确保选定的图像可以容纳在内存。点击[Create3D]创建卷。 </li><li>创建模块[SetWorldMatrix]，并设置下的“刻度”“初等变换”X，Y，Z适当的体素大小（0.6，0.7，1.0 10X 0.45NA目标）。链接[Compose3Dfrom2DFiles]至[SetWorldMatrix]。 </li><li>集矩阵和变换“基质组成”下“忽略”，“忽略”，“前进”。 </li><li>创建模块[Arithmetic1]，并设置“功能”，“反转（最大图）”。 链接[SetWorldMatrix]至[Arithmetic1]。 </li><li> [View3D创建模块和连接[Arithmetic1]。 </li><li> View3D，同时按下鼠标右键，移动鼠标左右调整门槛。您可以裁剪区域，改变方向（移动鼠标的同时按下鼠标左键），改变照明（体积rendering或MIP），开启/关闭背景等。 </li><li>见图。 10。 </li></ol></li></ol> 8。代表性的成果： 在这里，我们目前全脑的数据和细节。图。 12-15显示全脑墨汁，高尔基，和尼氏数据集1，4，3。 <img alt="图1" src="/files/ftp_upload/3248/3248fig1.jpg" /> 图1固定，染色和嵌入式的小鼠大脑 。 <img alt="图2" src="/files/ftp_upload/3248/3248fig2.jpg" /> 图2嵌入式鼠脑标本安装在试样环。 <img alt="图3" src="/files/ftp_upload/3248/3248fig3.jpg" /> 图3。刀刃扫描显微镜（KESM）。 KESM的照片显示标明其主要成分：（1）高速线扫描相机，（2）显微镜的物镜，（3）钻石刀大会和光准直器，（4）SPEcimen罐（水浸泡成像），（5）三轴高精度空气轴承阶段，（6）白光显微镜照明，（7）水泵去除切片组织（回），（8） PC服务器阶段控制和图像采集，（9）花岗岩基座，和（10）花岗岩桥。 <img alt="图4" src="/files/ftp_upload/3248/3248fig4.jpg" /> 图4。KESM的成像原理。 KESM操作主要是说明。目标和刀在地方举行，而标本的定位阶段动作（实线箭头），在分辨率为20 nm和旅行速度的1-5贴，并获得对钻石刀刮掉（5毫米宽10X的目标），生成一个薄片，流经刀（实线箭头）。行扫描成像是附近的刀很尖。 <img alt="图5" src="/files/ftp_upload/3248/3248fig5.jpg" /> 图5KESM相机和目标的重点控制。 <img alt="图6" src="/files/ftp_upload/3248/3248fig6.jpg" /> 图6。KESM刀大会和控制。 <img alt="图7" src="/files/ftp_upload/3248/3248fig7.jpg" /> 图7。初步通过观察口为重点。 <img alt="图8" src="/files/ftp_upload/3248/3248fig8.jpg" /> 图8。KESM级控制器应用程序（截图）。 <img alt="图9" src="/files/ftp_upload/3248/3248fig9.jpg" /> 图9。KESM栈处理器的应用程序（截图）。 <img alt="图10" src="/files/ftp_upload/3248/3248fig10.jpg" /> 图10。MeVisLab应用程序（截图）。 <img alt="图11" src="/files/ftp_upload/3248/3248fig11.jpg" /><br/&gt; 图11。KESM大脑图谱：Web界面（截图）。 <img alt="图12" src="/files/ftp_upload/3248/3248fig12.jpg" /> 图12。KESM全脑墨汁数据。 KESM数据栈卷可视化显示血管的数据集。 （ 一 ）特写血管的数据。宽度〜100 UM。 （BD）三个标准的意见，整个鼠脑血管（高分辨率数据的子采样）。宽度〜10mm的。 <img alt="图13" src="/files/ftp_upload/3248/3248fig13.jpg" /> 图13。KESM全脑鼠标高尔基数据。 <img alt="图14" src="/files/ftp_upload/3248/3248fig14.jpg" /> 图14。KESM高尔基数据的详细信息。 <img alt="图15" src="/files/ftp_upload/3248/3248fig15.jpg" /> 图15。KESM全脑尼氏数据。 （ 一 ）特写了NISSL数据。 〜300 UM 3。 （BD）三个标准的意见，整个鼠脑尼氏数据（高分辨率数据的子采样）。横截面所示的内部结构的一个明确的说法。

<ol>
	<li>Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+imaging+of+whole+small+animal+brains+with+the+knife-edge+scanning+microscope.">High-throughput imaging of whole small animal brains with the knife-edge scanning microscope.</a> Neuroscience Meeting Planner. Program No. 504.2, (2008).</li><li>Abbott, L. C., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+catecholaminergic+innervation+in+weaver+and+normal+mouse+cerebellar+cortices.">Ultrastructural analysis of catecholaminergic innervation in weaver and normal mouse cerebellar cortices.</a> Journal of Comparative Neurology. 426, 316-329 (2000).</li><li>Choe, Y., Abbott, L. C., Miller, D. E., Han, D., Yang, H. -. F., Chung, J. R., Sung, C., Mayerich, D., Kwon, J., Micheva, K., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+imaging,+analysis,+and+integration+of+mouse+brain+networks.">Multiscale imaging, analysis, and integration of mouse brain networks.</a> Neuroscience Meeting Planner. Program No. 516.3, (2010).</li><li>Choe, Y., Han, D., Huang, P. -. S., Keyser, J., Kwon, J., Mayerich, D., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+submicrometer+scans+of+mouse+brain+microstructure:+Neurons+and+vasculatures.">Complete submicrometer scans of mouse brain microstructure: Neurons and vasculatures.</a> Neuroscience Meeting Planner. Program No. 389.10, (2009).</li><li>Choe, Y., Abbott, L. C., Han, D., Huang, P. S., Keyser, J., Kwon, J., Mayerich, D., Melek, Z., McCormick, B. H., Ravi Rao, A., Cecchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy:+High-throughput+imaging+and+analysis+of+massive+volumes+of+biological+microstructures.">Knife-edge scanning microscopy: High-throughput imaging and analysis of massive volumes of biological microstructures.</a> High-Throughput Image Reconstruction and Analysis: Intelligent Microscopy Applications. , 11-37 (2008).</li><li>Choe, Y., Abbott, L. C., Ponte, G., Keyser, J., Kwon, J., Mayerich, D., Miller, D., Han, D., Grimaldi, A. M., Fiorito, F., Edelman, D. B., McKinstry, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+out+the+octopus+connectome+at+submicron+resolution+using+the+knife-edge+scanning+microscope.">Charting out the octopus connectome at submicron resolution using the knife-edge scanning microscope.</a> BMC Neuroscience. 11, 136-136 (2010).</li><li>Mayerich, D., Abbott, L. C., McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy+for+imaging+and+reconstruction+of+three-dimensional+anatomical+structures+of+the+mouse+brain.">Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain.</a> Journal of Microscopy. 231, 134-143 (2008).</li><li>Mayerich, D., Kwon, J., Sung, C., Abbott, L. C., Keyser, J., Choe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+macro-scale+Transmission+Imaging+of+Microvascular+Networks+Using+KESM.">Fast macro-scale Transmission Imaging of Microvascular Networks Using KESM.</a> Biomedical Optics Express. 2, 2888-2896 (2008).</li><li>McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Brain+Tissue+Scanner.">Development of the Brain Tissue Scanner.</a> Technical Report. , (2002).</li><li>Sporns, O., Tononi, G., K&#246;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+connectome:+A+structural+description+of+the+human+brain.">The human connectome: A structural description of the human brain.</a> PLoS Computational Biology. 1, 42-42 (2005).</li></ol>

托德霍夫曼是3Scan，一家商业公司，生产和销售的KESM技术（美国专利＃6744572）。

KESM允许了大量的生物标本（〜1厘米3）亚微米级的调查。这种卷到足以容纳整个小动物的器官，如大脑，肺，心脏，肾脏，，等等等器官的图像扫描，可以提供前所未有的定量信息，对这些器官的组织结构和启用各种功能的计算模型这些器官的各个方面，包括电路和网络动力学，电学性能，流体和空气流体动力学，以及肌肉动力学。 在这篇文章中详细的样品制备协议和事后分析协议，预计将有助于研究小组以外KESM产生的数据更容易获得。 KESM操作的协议将有助于这些外部研究人员欣赏这种独特的成像方式的好处和限制。

A correction was made to <a href="http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

A correction was made to <a href="http://http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

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

specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy

在高通量，高分辨率，三维显微技术的重大进展，使大量的亚微米分辨率的神经解剖数据的采集。刀口扫描显微镜（KESM）7，5，9，在作者的实验室开发和托管的生产规模的全脑数据的第一个这样的工具之一是。 KESM已被用于部分，并在亚微米分辨率的整体形象老鼠大脑，揭示神经元网络的错综复杂的细节（高尔基）1，4，8，血管网络（印度墨水）1，4，和细胞体内的分布（尼氏） 3 。 KESM使用不仅限于鼠标，也没有大脑。我们已经成功地成像章鱼大脑，小鼠肺，和大鼠脑组织。我们目前正在对整个斑马鱼的胚胎。这些数据可以极大地促进connectomics研究10，改善微循环和血流动力学研究;和体视学研究由provi丁一个确切的地面实况。 在这篇文章中，我们将描述的管道，包括样品制备（固定，染色，和嵌入），KESM配置和设置，切片和成像的KESM，图像处理，数据准备，数据可视化和分析。重点将样品制备和可视化/获得的KESM数据进行分析。我们期望在这篇文章中提出详细的协议，以帮助扩大的访问KESM，并提高其利用率。

Watch this Scientific Journal Video about Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy at JoVE.com

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

从大脑标本制备连续切片成像，用刀口扫描显微镜，数据可视化和分析的全过程描述。这项技术是目前用于收购鼠脑数据，但它是适用于其他器官，其他物种。

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 ...

specimen-preparation-imaging-analysis-protocols-for-knife-edge

Research

JoVE Journal

Bioengineering

17.5K 次观看.  Texas A&M University. 从大脑标本制备连续切片成像，用刀口扫描显微镜，数据可视化和分析的全过程描述。这项技术是目前用于收购鼠脑数据，但它是适用于其他器官，其他物种。

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

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 ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &amp;#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

混合μCT-FMT成像和图像分析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...