作者实验室的研究是由美国国立卫生赠款R37 - HL041026，R01 - HL086483和R01 - HL056786（SSS）和F32 - HL097463和T32 - AR048523由美国公共卫生服务（PB）研究院的支持。

1。动物护理和使用从体制上的动物护理和使用委员会的审查和批准后，至少12周龄雄性小鼠。鼠标是与戊巴比妥钠（60毫克/公斤）经腹腔（IP）注射麻醉。整个手术过程中和实验协议，麻醉维持补充剂（10-20％初始注射，IP）（每次30-60分钟;表示戒断反应到脚趾或尾巴捏）。实验过程完成后，鼠标过量巴比妥（IP）和颈椎脱位安乐死。 2。微量移液器和Microiontophoresis <ol><li>准备从高硼硅玻璃毛细管使用水平吸管拉马Microiontophoresis微量（内部尖端直径1微米）。我们有一个刚度〜5毫米锥度;不再锥更加灵活。 </li><li>微量移液器1M乙酰胆碱在18.2MΩ水溶解（Figure1A三）回填。进行回填，microcapillary管连接到0.2微米的过滤器加上一个注射器含利息激动剂（图1）。这些都可以随时编造（见下文），或从商业供应商购买。 microcapillary管插入后端的交付，同时撤回microcapillary管作为微管填充的微管和乙酰胆碱的解决办法是进入管腔。控股的微量用其尖端朝下，轻轻弹的微量气泡去除。 </li><li>微管是装在一个显微操纵器（图2）持有人担保。持有人有银线内的微连接的乙酰胆碱的解决方案，而这又是一个microiontophoresis程序员的正极连接到外部引脚。在组织编制的边缘安全的第二个银线连接到负极完成的电路是在准备到生理盐水先进的微尖。 </li><li>护电流应用，以防止血管扩张（或荧光反应）时，微管尖端的位置相邻的动脉壁。保留目前的微量提示（内部直径为1微米）和激动剂浓度的大小会有所不同，但定义的参数（我们使用1M乙酰胆碱，提示1微米〜200 nA的）的是高度重现。固定电流不再与目前通过电子触发图像采集发病同步（补充图1）弹射交付重合。 </li></ol> 3。显微<ol><li>铁磁不锈钢编造出一个为XY显微镜阶段的自定义平台。平台尺寸（1 / 8"× 12"× 13"），使有足够的空间，周围的磁基地（图2B），使定位为准备决定担保的期望（图2A）。Micromanipulators实验准备的位置micromanipulators。直接放置在制造平台周围感兴趣的地点（图2C）定位微量准备。 </li></ol> 4。代表性的成果<ol><li>随着微管尖端定位一个动脉旁，有荧光反应ACH（保留目前的调整，以防止乙酰胆碱泄漏）。低于阈值的刺激，有没有效果。然而，随着刺激强度的增加，内皮细胞钙荧光更远的距离，沿动脉逐步增加了，因为没有刺激的部位（图3）的荧光强度。 </li></ol><img src="/files/ftp_upload/2900/2900fig1.jpg" alt="图1" /> 图1。进行回填Microiontophoresis移液器的方法 。 A）microcapillary管（绿色箭头）是固定在一个压缩接头连接到一个0.2微米的过滤器（紫色箭头），然后附加到1M乙酰胆碱填补了注射器（蓝色箭头），B）microcapillary管喂进流离失所microiontophoresis通过吸管后端和解决方案，以填补管腔内的微管，C），一旦移液器填满，保持尖向下，轻轻一抖锥度以上，以去除气泡（黑色箭头）。 <img src="/files/ftp_upload/2900/2900fig2.jpg" alt="图2" /> 图2。微操作机器人放置自定义平台。 A）铁磁性的不锈钢平台被放置在一个自定义MVX10显微镜含有一个XY平移阶段的基本二）圆形磁性底座的紧凑型3轴micromanipulators的底部贴三）围绕定位的实验准备Micromanipulators（见相关的协议:朱庇特ID＃2874）的研究感兴趣的特定网站。 "红外紧凑的尺寸和多功能性，可以同时使用多种微量。 <img src="/files/ftp_upload/2900/2900fig3.jpg" alt="图3" /> 图3。在小动脉钙的荧光 。衬里的小动脉壁的内皮细胞的钙荧光增加刺激的强度。前3板的荧光反应的图像）250 NA，B）500 NA和C）1000 nA的弹射电流（500毫秒脉冲持续时间）。内皮细胞钙荧光增加的距离是在AC（〜130℃，270和400微米，分别）括号内表示。参考线横跨在每个面板动脉表示荧光反应（F / FO），乙酰胆碱刺激的部位。 D）F / FO随时间增加乙酰胆碱刺激的录音。弹射电流的增加，F / FO增加幅度和持续时间。请注意，100 nA的刺激阈值以下，并没有影响。 <img src="/files/ftp_upload/2900/2900sup1.jpg" alt="补充图1" /> 补充图1。电子触发电路原理图。这种电路是从一台个人计算机的并行端口相连。激活时，它提供了一个恒定的5V TTL脉冲。 7805芯片是一个5V稳压器连接到一个9 - 12V直流电源（电池一个合适的电压是罚款）。从7805的输出功率四路双向开关电路的输出。 1K的电阻两端的输入是从计算机。

<ol>
	<li>Majno, G., Palade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+inflammation.+1.+The+effect+of+histamine+and+serotonin+on+vascular+permeability:+an+electron+microscopic+study.">Studies on inflammation. 1. The effect of histamine and serotonin on vascular permeability: an electron microscopic study.</a> J. Biophys. Biochem. Cytol. 11, 571-605 (1961).</li><li>Majno, G., Palade, G. E., Schoefl, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+inflammation.+II.+The+site+of+action+of+histamine+and+serotonin+along+the+vascular+tree:+a+topographic+study.">Studies on inflammation. II. The site of action of histamine and serotonin along the vascular tree: a topographic study.</a> J. Biophys. Biochem. Cytol. 11, 607-626 (1961).</li><li>Grant, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Observation+of+Skeletal+Muscle+Blood+Vessels+(Rat+Cremaster).">Direct Observation of Skeletal Muscle Blood Vessels (Rat Cremaster).</a> J. Physiol. 172, 123-137 (1964).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc. Res. 5, 384-394 (1973).</li><li>Bohlen, H. G., Gore, R. W., Hutchins, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+microvascular+pressures+in+normal+and+spontaneously+hypertensive+rats.">Comparison of microvascular pressures in normal and spontaneously hypertensive rats.</a> Microvasc. Res. 13, 125-130 (1977).</li><li>Klitzman, B., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+hematocrit+and+red+cell+flow+in+resting+and+contracting+striated+muscle.">Microvascular hematocrit and red cell flow in resting and contracting striated muscle.</a> Am. J. Physiol. 237, 481-490 (1979).</li><li>Hungerford, J. E., Sessa, W. C., &amp;amp, S. e. g. a. l., S, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasomotor+control+in+arterioles+of+the+mouse+cremaster+muscle.">Vasomotor control in arterioles of the mouse cremaster muscle.</a> FASEB J. 14, 197-207 (2000).</li><li>Figueroa, X. F., Paul, D. L., Simon, A. M., Goodenough, D. A., Day, K. H., Damon, D. N., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+role+of+connexin40+in+the+propagation+of+electrically+activated+vasodilation+in+mouse+cremasteric+arterioles+in+vivo.">Central role of connexin40 in the propagation of electrically activated vasodilation in mouse cremasteric arterioles in vivo.</a> Circ. Res. 92, 793-800 (2003).</li><li>Wolfle, S. E., Schmidt, V. J., Hoepfl, B., Gebert, A., Alcolea, S., Gros, D., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin45+cannot+replace+the+function+of+connexin40+in+conducting+endothelium-dependent+dilations+along+arterioles.">Connexin45 cannot replace the function of connexin40 in conducting endothelium-dependent dilations along arterioles.</a> Circ. Res. 101, 292-1299 (2007).</li><li>Milkau, M., Kohler, R., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+importance+of+the+endothelial+K++channel+SK3+and+connexin40+in+arteriolar+dilations+during+skeletal+muscle+contraction.">Crucial importance of the endothelial K+ channel SK3 and connexin40 in arteriolar dilations during skeletal muscle contraction.</a> FASEB J. 24, 3572-3579 (2010).</li><li>Bagher, P., Duan, D., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+impaired+neurovascular+transmission+in+a+murine+model+of+Duchenne+Muscular+Dystrophy.">Evidence for impaired neurovascular transmission in a murine model of Duchenne Muscular Dystrophy.</a> J. Appl. Physiol. 110, 601-610 (2011).</li><li>Tallini, Y. N., Brekke, J. F., Shui, B., Doran, R., Hwang, S. M., Nakai, J., Salama, G., Segal, S. S., Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagated+endothelial+Ca2++waves+and+arteriolar+dilation+in+vivo:+measurements+in+Cx40BAC-GCaMP2+transgenic+mice.">Propagated endothelial Ca2+ waves and arteriolar dilation in vivo: measurements in Cx40BAC-GCaMP2 transgenic mice.</a> Circ. Res. 101, 1300-1309 (2007).</li><li>Grant, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+denervation+on+skeletal+muscle+blood+vessels+(rat+cremaster).">The effects of denervation on skeletal muscle blood vessels (rat cremaster).</a> J. Anat. 100, 305-316 (1966).</li><li>Proctor, K. G., Busija, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+among+arteriolar,+regional,+and+whole+organ+blood+flow+in+cremaster+muscle.">Relationships among arteriolar, regional, and whole organ blood flow in cremaster muscle.</a> Am. J. Physiol. 249, H34-H41 (1985).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+blood+flow+in+the+microcirculation:+Role+of+conducted+vasodilation.">Regulation of blood flow in the microcirculation: Role of conducted vasodilation.</a> Acta Physiol. , (2011).</li><li>Hill, M. A., Simpson, B. E., Meininger, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cremaster+muscle+hemodynamics+due+to+disruption+of+the+deferential+feed+vessels.">Altered cremaster muscle hemodynamics due to disruption of the deferential feed vessels.</a> Microvasc. Res. 39, 349-363 (1990).</li></ol>

所有的程序和协议，涉及动物，美国密苏里大学动物护理和使用委员会的批准，并符合美国国立卫生研究院实验动物的照顾和使用指南进行生产这篇文章是由斯坦福大学光电赞助。

这里所描述的协议表明准备和实施microiontophoresis微量的方法。乙酰胆碱的交付是用来说明在麻醉小鼠的动脉钙信号基本内皮依赖性血管舒张功能。我们的结果表明，乙酰胆碱增加microiontophoresis微量电流（图3）弹射力度内皮细胞钙荧光增加距离。乙酰胆碱缺乏休息的条件下的荧光增加，表示可以忽略不计，从微渗漏。缺乏应对降至100 nA刺激的强度（图3，传说）说明，内皮细胞钙增加所需的刺激阈值强度。这些技术可以很容易地适应其他血管活性药物和组织准备。 实际考虑:microiontophoresis工作研究动脉反应，几件事情应该得到承认。虽然它已被证明难以确定实际交付激动剂浓度，刺激 - 反应曲线之内和之间的准备重现。这些都可以通过脉冲持续时间不变（例如，500毫秒）和不同的弹射电流（例如，250，500和1000 NA;图3）。此外，弹射目前可以保持不变（例如，500 NA）和脉冲持续时间不同（例如，250，500和1000毫秒）。对于定义的激动剂浓度的行动，准备可以灌流合适的解决方案5。由于溶质的弹射动力，是电荷的运动，要交付的代理人必须携带的净电荷从微流离失所。为了确保代理人的利益是主要的电荷载体，它溶解在高浓度（例如，对乙酰胆碱的1男），以尽量减少电渗。当这需要操纵微量填充液的pH值，车辆的控制规定，以确定任何非特异性影响。适当的控制，也应单独通过的电流进行（例如，用充满生理盐水的微量）。代理其网站发布的扩散的有效距离应该是确定的，是最容易消失的一种生理反应（如血管扩张，或在细胞内钙离子的乙酰胆碱后弹射上升）由经验的微量定位于定义目标站点的距离。在实践中，有效扩散距离是很大的影响，如何定位在组织的微尖;例如，如果压入的组织和其尖端的技巧是闭塞，比弹射受损。过度的结缔组织是特别麻烦，应该从组织表面除去，在手术准备。也应注意给消耗的指定代理商的一角的可能性。这是尽量减少使用相对短的脉冲（例如，≤1秒）。持续电流（例如，几秒钟），激动剂可能被开除的提示更迅速地超过它可以从散装油中的微解决方案扩散所取代。 因为微不足道的体积是流离失所与microiontophoresis，如果之一是试图以改变的地方离子环境（例如，以提供一个极化K +刺激），可以有效地实现与microiontophoresis但是很容易的压力散装液体所需的弹射实现组成。一个动脉的局部刺激，内部直径为2-3微米的微管提示的工作以及与弹射压力4-5磅（28-35千帕）和脉冲持续时间（如1秒）控制一个电磁阀10。持续提供从微量到微血管代理是必需的，最好是使用适当的内部尖端直径（例如，〜10微米）11,12微量压力弹射。静水列的已知与活塞阀的高度提供了一种廉价的，良好定义的恒定压头开/关。流速由枪头和驾驶压力内径。一如往常，车辆控制是必不可少的，以排除非特异性的压力弹射行动。

<table><tbody><tr><td> 使用材料 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>硼硅玻璃毛细管</td><td>华纳仪器</td><td> GC120F - 10 </td><td></td></tr><tr><td>卧式移液器拖轮</td><td>萨特仪器</td><td>模型P - 97 </td><td></td></tr><tr><td>氯化乙酰胆碱</td><td> Sigma - Aldrich公司</td><td> A6625 </td><td></td></tr><tr><td>露儿枢纽适配器</td><td> Martech </td><td> AC1343 </td><td>为了确保microcapillary管</td></tr><tr><td> 0.2微米尼龙泰坦过滤器</td><td>孙斯里兰卡</td><td> 42204 - NN </td><td>低保留体积，以减少损失</td></tr><tr><td> 5 ml注射器</td><td>流式细胞 - 迪金森公司</td><td> 309603 </td><td></td></tr><tr><td>移液器架</td><td>华纳仪器</td><td> E45W - M12VH </td><td></td></tr><tr><td>银线</td><td>华纳仪器</td><td> AG10W </td><td> 0.25毫米直径</td></tr><tr><td> 3轴显微</td><td>锡斯基尤设计仪器</td><td> DT3 - 100，MXB，MXC，MGB / 8 </td><td>机械臂组件，如图所示</td></tr><tr><td> microiontophoresis当前的编程</td><td>世界精密仪器</td><td>型号260 </td><td></td></tr><tr><td>触发装置</td><td>自定义</td><td>自定义</td><td>电路提供增刊。图1 </td></tr><tr><td>不锈钢板</td><td>麦克马斯特 - 卡尔</td><td> 1 / 8"× 12"× 12" </td><td>根据设计</td></tr><tr><td>强化数码相机</td><td>斯坦福大学光电公司</td><td> XR/Mega-10 </td><td>派控制软件集成</td></tr></tbody></table>

microiontophoresis and micromanipulation for intravital fluorescence imaging of the microcirculation

Microiontophoresis需要通过微管尖端的电流通过溶质在指定的网站内，提供一个实验的准备。 Microiontophoresis可以模拟提供到神经元再现2的神经递质和神经肽的突触传递的 1 。可以忽略不计容积（液）的位移，避免了机械扰动的实验准备。改善微循环3适应这些技术，使血管扩张和血管收缩的机制，在微观层面，在 体内 4,5的研究。一个关键的优势是对等的本地化交付使血管舒缩研究没有唤起全身或自反血压和组织血流量的变化，从而揭示了内在属性的微血管内微血管网络的定义站点的反应。 的microiontophoresis限制，传递到感兴趣的网站的代理的精确浓度是难以确定6。然而，其释放的微量提示是成正比的强度和持续时间的弹射目前2,7，这样，重复性的刺激-反应关系可以很容易确定的实验条件下定义（如下所述） 。影响微电泳交付的其他因素包括其在溶液中的溶质浓度和电离。内部的微管尖端直径应〜1微米或更小，以尽量减少扩散的"漏"，可以用护电流抵消。因此，向外（正）目前用于弹出保留内微阳离子和负电流。 微量的制备是促进了先进的电子车夫。微量移液器含有灯丝，玻璃毛细管拉成的微尖的"芯"的解决方案，从后端（"回填"）填补。这样做是通过插入microcapillary管连接注射器的微腔的兴趣和退出的解决方案解决方案。 Micromanipulators启用所需的微量安置在实验的准备。可以放置一个可移动的基础上安装Micromanipulators各地的准备，根据地形的微血管网络（下文）。 本议定书演示到一个鼠标提睾肌准备的动脉（见相关的协议:朱庇特的ID＃2874），产生内皮依赖性血管舒张的乙酰胆碱microiontophoresis（乙酰胆碱+ CL - ）。刺激同步交付使用电子触发与数字化图像采集。 Cx40 BAC - GCaMP2转基因小鼠9的使用，使细胞内钙离子反应，在生活改善微循环的底层在动脉内皮细胞的血管舒张的可视化。

Watch this Scientific Journal Video about Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation at JoVE.com

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis需要在电势之间的内部和外界的微的差异，从一个微离子的运动。生物活性分子，从而交付的电流成比例。我们说明结合显微研究在微循环内皮依赖性血管舒张的乙酰胆碱microiontophoresis。

Microiontophoresis和显微操作的微循环活体荧光成像

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

microiontophoresis-micromanipulation-for-intravital-fluorescence

Research

JoVE Journal

Biology

14.4K 次观看.  University of Missouri. Microiontophoresis需要在电势之间的内部和外界的微的差异，从一个微离子的运动。生物活性分子，从而交付的电流成比例。我们说明结合显微研究在微循环内皮依赖性血管舒张的乙酰胆碱microiontophoresis。

视频: Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

生于正常化荧光整个心脏成像的光学投影层析成像

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

科研

生物学

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

鼠标提睾肌肌肉制备的微循环活体成像

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

衣藻中心粒单粒子重建的分离与荧光成像

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...