这项研究部分经费由美国国立卫生研究院，授予R01 EB007594，国防部乳腺癌研究计划署的建议BCO74699P7，和苏珊科曼基金会授予98188972分之26152。

1。 Microendoscope大会这里描述（图1a）高分辨率microendoscope应被视为一个基本配置与几个变化大会和应用可能。我们描述在这里详细体现的平台，其目的是作为一种荧光造影剂与proflavine。 Proflavine是一个明亮的核与445 nm和515 nm处的吸收峰和发射波长分别染色。其他造影剂的使用，将要求用户选择适当激发，发射和二向色滤光片。高分辨率microendoscope的几个要素是通用的，可从多个供应商获得。例如，光学机械定位元件Thorlabs，新港，利诺斯等等。紧凑型的CCD相机都可以从公司的Point Grey研究，Prosilica，并Retiga;摄像机的灵敏度，应考虑到要使用的荧光团的亮度，以及所需的帧速率的选择。高功率发光二极管（LED）可能获得LUXEON，克里和Nichia等。住友，藤仓，和肖特光纤束。在选择特定的应用程序组件，用户应该考虑参与荧光团的浓度之间荧光显微镜的内在关系，漂白，照度，摄像机的灵敏度，增益，曝光时间。 <ol><li> 6“笼棒连接1.5”笼棒，形成了7.5“长棒对螺丝拧入倍镜单位面对这些棒。0.5”到对面的脸棒。滑动到笼笼棒多维数据集，通过这两个通孔。 2“棒旋入笼立方体（图1b）面朝。 </li><li>使用C型安装的SM1适配器将相机安装在一个笼子里板。安全笼0.5“笼棒板，面对倍镜单位冲洗。 </li><li> 3“的长镜头管插入”管“的镜头和安全护圈与镜头焦距的镜头。应选择光纤束的核心是由至少两个像素采样时预计相机上。拖放到管排放过滤器第一扣环上，并添加另一个环，以确保在过滤器。观察过滤器的方向公约表示过滤器的制造商。侧旋入镜头管笼多维数据集最近的CCD相机。注意，在图1c，这个镜头和过滤器没有3“镜头清晰管。 </li><li>将相机连接到计算机，并查看屏幕上的图像。在一个遥远的对象直接的笼大会和笼沿导轨滑动立方体，直到图像出现在焦点。锁定在此位置的笼子立方体。 （这是一个简单的方法，确保管结合的无限远校正物镜镜头时，将形成一个集中的形象）。 </li><li>插入到持有人，并在笼子里立方体在45 °的分色镜。 </li><li>螺杆的物镜，通过一个SM1应当在螺纹接头RMS和一个可调的透镜筒，到对面的腾笼换管镜架立方体面前。一个Z -翻译可调透镜筒可用于更容易聚焦。螺杆进笼子板的SMA连接器，安装在棒大约在物镜的工作距离，。 </li><li>装入一个LED在一个笼子里的钢板和幻灯片上的2月底的“棒添加一个镜头，0.5”镜头，当这个管拧入笼立方体的一面，它会形成一个形象的LED管等。充满物镜后孔径。 （科勒照明）配置将确保光纤束的近端面均匀照明。激发滤光片0.5“透镜筒和地方安全锁紧环（图1C）。 </li><li>一个SMA连接器连接到一个光纤束。旋入装上笼棒的SMA插座的SMA连接器捆绑。直接对一个宽带光源（荧光灯照明就足够了）束末端，并观察对CCD相机的捆绑近端面对的形象。调整物镜的位置，拧紧或笼立方体，直到纤维束图像集中出现。个人的核心应该是清晰可见（图2）。 </li></ol> 2。 GRIN透镜大会 microendoscope的空间分辨率可以增加附加一个微型镜头或镜头装配的纤维束的远端提示。这些光学配置，而不​​是直接放置在组织束尖端，尖端demagnification组织表面成像上，从而提高空间采样频率的光的GUID规定ING核心的纤维束。 demagnification程度对应在空间分辨率，并在同一时间的增加，按比例减少在现场的。梯度折射率（GRIN）透镜组件与光纤兼容GrinTech，核供应国集团，肖特，等等，可以直接粘在纤维束的远端提示。 <ol><li>为您的应用程序所需的放大倍率和工作距离选择一个GRIN透镜。确保镜片直径超过计划与纤维束。我们建议，独立的3轴手动定位阶段下安装一个低功率的显微镜或立体精确对准前粘接的纤维束和GRIN透镜。 </li><li>光学胶（如诺兰紫外光固化胶粘剂）上放置一个镜头或捆绑面对下降。使用手动定位，接触到的两个组成部分。接口暴露在紫外灯制造商推荐的剂量。 </li><li>为了保护GRIN透镜和保税接口，铝毛细管（含小零件）的短长，可以用附上的联合。滑过油管与环氧地方联合和安全。热缩管，可用于完成大会。 </li></ol> 3。 Microendoscope成像<ol><li>应用造影剂成像的细胞或组织。 proflavine（0.01％W / V在PBS）， 在体外培养细胞成像，可以进行短暂的潜伏期（&lt;1分钟），并彻底冲洗。 体外组织标本或体内组织的成像是可能以下的染料局部应用。 proflavine摄取这些条件下成像可能在几秒钟内，并持续几分钟，被认为是近乎瞬时的。 </li><li>将光纤束光成像样品接触。当成像细胞文化或体外组织标本中，我们建议安装在成像过程中的稳定性XYZ轴手动定位阶段的安全夹具纤维束的末端。 </li></ol> 4。代表性的成果： 正确组装时，microendoscope将落射荧光显微镜，通过一个连贯的光纤束中继。为了达到最佳的成像效果，应引起重视，以确保满足三个关键条件： <ol><li>纤维束的近端面对应无散焦到CCD相机的成像，以实现系统的全部决议。图2A，B，C表现出部分的纤维束成像差的焦点，稍有失焦，和良好的重点，分别。找到最佳的重点是通过调整物镜相捆绑面对的轴向位置。 </li><li>纤维束的近端表面应均匀地照亮在其整个直径（实地查看）。正如议定书案文（1.7）中所述，这是通过配置科勒照明的照明光学系统。图2d显示亮起时获得一个统一的荧光样品在这个首选的配置，展示了关键的照明下的相应的结果与图2E，图像。在后一种情况下，LED的结构成像上的纤维束面，在这个多余的图案叠加后的真实样品的结构外观。 </li><li>纤维束的近端和远端的面孔应保持清洁，无划痕和芯片。图2F演示捆绑面对的碎片的存在，破坏纤维周边5时许在小芯片的形式。以同样的方式与传统的光纤连接器清洁，用镜头纸，异丙醇，或标准的光纤清洁工具，杂物可从捆绑面临。如果纤维束两端被划伤或插话，或如果束沿其长度打破，面对可能被磨光平标准手册，或机械抛光技术。我们建议12-15微米为一个初步的粗抛光研磨纸，用0.5-1.0微米纸最后的抛光。 </li></ol>图3a显示1483细胞在体外成像，下面的标签与proflavine和光裸纤维束对样品的位置。图3b显示在空间分辨率和减少由一个2.5倍的笑容保税束尖端镜头提供的字段的视图改善。电影1表明，在活体成像的小鼠模型的乳腺脂肪垫。在这里，一个0.5毫米的外径（330微米领域的视图）的纤维束是通过一个21号针头和先进的组织。脂肪细胞清晰可见，由于心动周期明显此次收购的议案15帧每秒。图3c演示成像的在一个健康的人类志愿者的口腔粘膜，这个时候，使用1.5毫米外径（1.4毫米场的视图）一个较大的纤维束。在所有显示的例子，proflavine被用作核标签荧光造影剂。 <img src="/files/ftp_upload/2306/2306fig1.jpg" alt="图1" /> 图1。装配高分辨率microendoscope（HRME）。 （一）该HRME系统的示意图。 （b）大会的主要光学机械支撑结构。 （三）添加光学元件，照明LED，和CCD相机。 （四）照片HRME系统，封装在一个10“× 8”X 2.5“圈地。 <img src="/files/ftp_upload/2306/2306fig2.jpg" alt="图2" /> 图2。成立HRME。成像的例子，与光纤束（一）贫困重点，（b）关闭好重点，（C）理想的重点。 （四），科勒（统一）照明下一个统一的目标bundle的远端提示荧光成像。 （E）一个统一的荧光目标成像关键的照明下，与源对象的结构明显。 （F）松散的组织和细胞，能坚持到纤维束的脸，这是也很容易出现轻微损坏，在其周边。 <img src="/files/ftp_upload/2306/2306fig3.jpg" alt="图3" /> 图3。HRME的成像。 （一）1483细胞， 在体外成像proflavine 0.01％（W / V）的标签后的裸纤维束（IGN-08/30 ）。 （二）相同的1483细胞培养（一）所示，与纤维束连接与2.5倍GRIN透镜成像。 （三） 在体内正常人体口腔粘膜图像，外敷proflavine 0.01％（W / V）。 电影1。成像乳腺脂肪垫鼠标，通过插入传递到组织中的21号针管腔内外径450微米的纤维束。 Proflavine 0.01％（W / V）被送到成像网站通过同一个针头插入成像纤维前。 <a href="http://www.jove.com/files/ftp_upload/2306/5-11-1 2306_Pierce_movie1.avi">点击这里观看视频</a>

<ol>
	<li>Pierce, M. C., Javier, D. J., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+contrast+agents+and+imaging+systems+for+detection+and+diagnosis+of+cancer.">Optical contrast agents and imaging systems for detection and diagnosis of cancer.</a> Int. J. Cancer. 123, 1979-1990 (2008).</li><li>Muldoon, T. J., Pierce, M. C., Nida, D. L., Williams, M. D., Gillenwater, A., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular-resolution+molecular+imaging+within+living+tissue+by+fiber+microendoscopy.">Subcellular-resolution molecular imaging within living tissue by fiber microendoscopy.</a> Opt. Express. 15, 16413-16423 (2007).</li><li>Muldoon, T. J., Anandasabapathy, S., Maru, D., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+in+Barrett's+esophagus:+a+novel,+low-cost+endoscopic+microscope.">High-resolution imaging in Barrett's esophagus: a novel, low-cost endoscopic microscope.</a> Gastrointest. Endosc. 68, 737-744 (2008).</li><li>Muldoon, T. J., Thekkek, N., Roblyer, D., Maru, D., Harpaz, N., Potack, J., Anandasabapathy, S., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+quantitative+image+analysis+criteria+for+the+high-resolution+microendoscopic+detection+of+neoplasia+in+Barrett's+esophagus.">Evaluation of quantitative image analysis criteria for the high-resolution microendoscopic detection of neoplasia in Barrett's esophagus.</a> J. Biomed. Opt. 15, 026027-026027 (2010).</li><li>Zhong, W., Celli, J. P., Rizvi, I., Mai, Z., Spring, B. Q., Yun, S. H., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+high-resolution+fluorescence+microendoscopy+for+ovarian+cancer+detection+and+treatment+monitoring.">In vivo high-resolution fluorescence microendoscopy for ovarian cancer detection and treatment monitoring.</a> Br. J. Cancer. 101, 2015-2022 (2009).</li><li>Dubaj, V., Mazzolini, A., Wood, A., Harris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+fibre+bundle+contact+imaging+probe+employing+a+laser+scanning+confocal+microscope.">Optic fibre bundle contact imaging probe employing a laser scanning confocal microscope.</a> J. Microsc. 207, 108-117 (2002).</li><li>Dromard, T., Ravaine, V., Ravaine, S., L&#233;v&#234;que, J. -. L., Sojic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+in+vivo+imaging+of+human+skin+corneocytes+by+means+of+an+optical+fiber+bundle.">Remote in vivo imaging of human skin corneocytes by means of an optical fiber bundle.</a> Rev. Sci. Inst. 78, 053709-05 (2007).</li><li>Udovich, J. A., Kirkpatrick, N. D., Kano, A., Tanbakuchi, A., Utzinger, U., Gmitro, A. F. <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> Appl. Opt. 47, 4560-4568 (2008).</li><li>Barretto, R. P. J., Messerschmidt, B., Schnitzer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fluorescence+imaging+with+high-resolution+microlenses.">In vivo fluorescence imaging with high-resolution microlenses.</a> Nat. Methods. 6, 511-512 (2009).</li><li>Rouse, A. R., Kano, A., Udovich, J. A., Kroto, S. M., Gmitro, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+demonstration+of+a+miniature+catheter+for+a+confocal+microendoscope.">Design and demonstration of a miniature catheter for a confocal microendoscope.</a> Appl. Opt. 43, 5763-5771 (2004).</li><li>Kester, R. T., Christenson, T., Richards-Kortum, R., Tkaczyk, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+cost,+high+performance,+self-aligning+miniature+optical+systems.">Low cost, high performance, self-aligning miniature optical systems.</a> Appl. Opt. 48, 3375-3384 (2009).</li></ol>

MP和DY都没有透露。 RRK拥有的专利与显微成像平台。

高分辨率内窥镜技术的描述，这里提供了一个可视化细胞原位细节的灵活，功能强大，和成本效益的方法基本的生物医学和临床研究领域的研究人员。组装成像系统，我们已经描述了一个协议， 在体外细胞培养表明其使用，并在动物和人体组织在体内。虽然这里介绍的成像结果proflavine作为荧光造影剂，与其他群体已经证明LED照明波长选择5-7其他染料的激发/发射光谱相匹配的过滤器系统的版本。 分辨率和领域的初步确定由核心到核心的间距和成像光纤束的直径。我们已经使用了约4微米的间距，芯芯和成像直径330微米（1电影），720微米（图2，图3A，B），和1400微米（图3c）捆绑。小束可以通过较窄的表针，大大超过弹性较大的纤维。我们和其他8，在某些情况下，注意到从纤维的自体荧光排放外观包本身。当试图在紫外线波长激发的荧光，或在红色光谱范围内收集排放，应注意的整体被测信号的自体荧光纤维束水平。 虽然大部分高清晰度内窥镜的工作报告，至今已经使用了裸纤维束，可提供进一步放大，利用保税远端尖的梯度折射率镜片。梯度折射率透镜提供一个简单和经济的方式，以增加空间分辨率，虽然是众所公认的易感性光学像差和有限娜。如果GRIN透镜的性能不足为特定的应用程序，混合GRIN / 9球面镜片目标或微型物镜组件 10-11可以采用。 高分辨率microendoscope这里描述的基本上为宽视场落射荧光显微镜，因此没有光学切片（如共聚焦或非线性显微镜）是可以预料的。根据我们的经验，用455 nm激发光作为造影剂的外用proflavine，光线主要是收集相应的几个细胞层的深度。 这个协议应该使读者组装台式高分辨率microendoscope，紧凑的面积为10“× 8”。如果需要的话，系统可能会被封闭在一个盒子里，由电池供电（图1d）和电气元件（LED和摄像头）。许多紧凑型相机可以通过IEEE - 1394（火线）和计算机主机的USB端口供电。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CCD camera</td>
<td>&nbsp;</td>
<td>Point Grey Research</td>
<td>GRAS-14S5M</td>
<td>&nbsp;</td>
<tr>
<td>LED</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>M455L2</td>
<td>Selected for use with proflavine &#x2013; other fluorophores may require different parts</td>
</tr>
<tr>
<td>Excitation filter</td>
<td>&nbsp;</td>
<td>Semrock</td>
<td>452/45</td>
<td>Selected for use with proflavine &#x2013; other fluorophores may require different parts</td>
</tr>
<tr>
<td>Emission filter</td>
<td>&nbsp;</td>
<td>Semrock</td>
<td>550/88</td>
<td>Selected for use with proflavine &#x2013; other fluorophores may require different parts</td>
</tr>
<tr>
<td>Dichroic mirror</td>
<td>&nbsp;</td>
<td>Chroma</td>
<td>485 DCLP</td>
<td>Selected for use with proflavine &#x2013; other fluorophores may require different parts</td>
</tr>
<tr>
<td>Objective lens</td>
<td>&nbsp;</td>
<td>Thorlabs (Olympus)</td>
<td>RMS 10X</td>
<td>&nbsp;</td>
<tr>
<td>Tube lens</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>AC-254-150-A1</td>
<td>Select focal length to achieve required magnification to CCD</td>
</tr>
<tr>
<td>Condenser lens</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>ACL2520</td>
<td>&nbsp;</td>
<tr>
<td>Cage cube unit</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>C6W, B1C, B3C, B5C, SM1CP2</td>
<td>&nbsp;</td>
<tr>
<td>Cage rods and plates</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>ER05 (x4), ER1.5 (x2), ER2 (x2), ER6 (x2), CP02 (x3)</td>
<td>&nbsp;</td>
<tr>
<td>Fold mirror unit</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>KCB1, PF10-03-G01</td>
<td>&nbsp;</td>
<tr>
<td>Lens tubes</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>SM1L05, SM1L30, SM1V05 (or SM1Z)</td>
<td>&nbsp;</td>
<tr>
<td>Adapters / couplers</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>SM1A3, SM1A9, SM1T2 (x2)</td>
<td>&nbsp;</td>
<tr>
<td>SMA connectors</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>SM1SMA, 11040A</td>
<td>&nbsp;</td>
<tr>
<td>LED driver</td>
<td>&nbsp;</td>
<td>Thorlabs</td>
<td>LEDD1B TPS001</td>
<td>&nbsp;</td>
<tr>
<td>Fiber optic bundle</td>
<td>&nbsp;</td>
<td>Sumitomo</td>
<td>IGN-08/30</td>
<td>Larger or smaller bundles are available (Sumitomo / Fujikura)</td>
</tr>		</tbody>
		</table>
		</div>

high-resolution fiber-optic microendoscopy for in situ cellular imaging

许多生物和临床研究，细胞水平分辨率的形态和功能的纵向研究和分析。传统上，并行运行多个实验，研究进行评估的连续时间点，通过光镜移除的个别样本。已经开发了几个活体技术，共聚焦，多光子和二次谐波显微成像能力在 原地 1展示。然而，有了这些系统，所需的基础设施是复杂和昂贵，涉及激光扫描系统和复杂的光源。在这里，我们现在可以在一天建成使用的，现成的5000美元以下的组件的设计和一个高分辨率的microendoscope大会​​的协议。该平台提供了灵活性，在图像分辨率方面，场的观点，工作波长，而我们描述了如何将这些参数可以很容易地修改，以满足最终用户的特定需求。 我们和其他人探讨高分辨率microendoscope（HRME） 在体外细胞培养2-5使用，在切除6和活生生的动物组织2,5和2,7 体内的人体组织。用户已经报告了几个不同的荧光造影剂的使用，包括proflavine 2-4苯并卟啉衍生的单酸环A（BPD - MA）5，和fluoroscein 6,7，所有这些都得到了充分的，或研究从美国FDA的批准，在人类受试者使用。高分辨率内窥镜，在这里描述的形式，可以上诉到一个广泛的基础和临床科学的研究人员。该技术提供了一个有效和经济的的方法的补充，使用户能够执行高解析度，在原地纵向成像传统的台式显微镜， 。

Watch this Scientific Journal Video about High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging at JoVE.com

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

在许多生物学和临床的情况下是有利的，以研究细胞过程，因为他们在家乡的微环境演变。在这里，我们描述了一个低成本的光纤显微镜，细胞培养，动物实验和临床病人的研究中可以提供实时成像的组装和使用。

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.  ...

high-resolution-fiber-optic-microendoscopy-for-situ-cellular

Research

JoVE Journal

Bioengineering

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

34.3K 次观看.  Rice University. 在许多生物学和临床的情况下是有利的，以研究细胞过程，因为他们在家乡的微环境演变。在这里，我们描述了一个低成本的光纤显微镜，细胞培养，动物实验和临床病人的研究中可以提供实时成像的组装和使用。

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

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

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 ...

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

在芽殖酵母连续高分辨率显微观察复制性衰老

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

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 ...

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 ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

用原位滞后测量进行磁畴成像的优化设置和协议

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

人类胰岛神经网络的高分辨率3D 成像

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct&#8212;pDawn-Ag43&#8212;that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with&#160;pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 &#956;m can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.

We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

使用 pDawn-Ag43 的高分辨率图案生物膜沉积

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms ...