这项工作是由美国国立卫生研究院批准安永14990支持。

第1部分：夹层和双极细胞的分离<ol><li>准备在表2中列出的解决方案;铃声“（外部）解决方案的pH值应调整到7.4用NaOH和内部解决方案的pH值应调整至7.2与CsOH。光用铝箔保护内部的解决方案，并保持在4 °，直到使用C; </li><li>暗适应至少30分钟前清扫金鱼; </li><li>而动物黑暗的适应，准备5毫升的透明质酸酶（V型透明质酸酶1100单位/毫升在低Ca 2 +林格;西格玛，圣路易斯，密苏里州）和10毫升的L -半胱氨酸（0.5毫克/毫升在低Ca 2 +林格）的解决方案，并权衡木瓜蛋白酶（冻干粉，40个单位/毫升;消化溶液5毫升，圣路易斯，密苏里州） ; </li><li>安乐死手术剪刀快速断头金鱼，破坏大脑和脊髓＃11手术刀刀片; </li><li>删除摧毁＃7弯曲杜蒙钳的帮助下，眼外肌和虹膜剪刀切割视神经眼睛; </li><li>一块滤纸和穿刺＃11手术刀刀片的一角巩膜角膜缘上放置一个灯泡眼; </li><li>介绍了一双内刺破整体vannas剪刀的刀片和削减整个眼前段路程; </li><li>其余的视杯上放置一个小块滤纸，并施加一定的压力，以纸浸泡玻璃体; </li><li>电梯与视网膜连接到它的滤纸和削减vannas剪刀一样的视神经; </li><li>将滤纸含有35毫米的塑料培养皿用透明质酸酶溶液和关闭＃7杜蒙镊子帮助滤纸的视网膜剥离的视网膜; </li><li>视网膜一半的工业碳素钢单刃刀片切成4-6块，让它坐在在20分钟的透明质酸解决方案; </li><li>虽然等待透明质酸生效，添加5毫升的L -半胱氨酸的解决方案，以木瓜，让坐下，直到液体变成透明（约5-10分钟）; </li><li>在较低的Ca 2 +林格洗净视网膜3X件，并让他们坐30-35分钟，在木瓜蛋白酶的解决方案; </li><li>洗净低的Ca 2 +林格视网膜3X件和存储，直到4 ° 35毫米的塑料培养含有低Ca 2盘的C +林格; </li><li>游离的细胞，把一块视网膜中含有微量低的Ca 2 +林格氏液500毫升管，并慢慢地磨碎用玻璃离解吸管吹打向上和向下的视网膜上，仔细不产生任何气泡。本生灯加热的玻璃巴斯德吸管尖，稍微弯曲解剖钳的帮助下解离吸液管制造; </li><li>板分离出的细胞，加入了视网膜悬挂下降一个自制的录音室，以前用2 毫升低 Ca 2 +林格充满。分庭由在中间的圆形整个一个35毫米的塑料培养皿和1.78折射率玻璃底部粘用硅橡胶（Sylgard 184（德国PlanOptik，）的圆形盖玻片的下半部分;道康宁米德兰，MI）。 </li></ol>第2部分：双极细胞装载和洗出 TIRF成像的突触小泡是最好的开展具有非常高的NA目标和敏感的相机使用客观型TIRFM显微镜。对于我们的实验中，我们选择使用一个1.65 NA目标（APO X100 Ø人力资源，不适用1.65，奥林巴斯，日本）与EMCCD（级联512B，罗珀科学，图森，AZ）。使用非常高NA的目标，就必须使用高折射玻璃盖玻片和浸入液（含硫量DI -甲基碘）。在我们的条件，激发光指数衰减的领域是有限的，长度约50 nm的不断。 <ol><li>添加高折射率液体（M系列，折射率= 1.7800，Cargille实验室，雪松树丛，新泽西州）的下降显微镜物镜; </li><li>仔细将录音室，显微镜物镜上，精心安装接地电极和灌流出口管道，以室; </li><li>让室坐10-20分钟，显微镜，让细胞下沉和坚持的底部; </li><li>在此期间，准备为1mm Trolox的® ((±)- 6 -羟基- 2 ,5,7,8 - tetramethylchromane - 2 -羧基酸5毫升; Sigma公司，圣路易斯，密苏里州）的解决方案在高K +林格。超声，直至溶解; </li><li>准备15毫升ADVASEP - 7洗涤液：1毫米ADVASEP - 7（Sigma公司，圣路易斯，密苏里州）在低Ca 2 +林格。请注意，ADVASEP - 7的使用是可选的，如果需要的话，可以省略; </li><li>清除的灌流李NES，并添加ADVASEP - 7洗涤液，低Ca 2 +林格和控制振铃的灌流系统; </li><li>拉加载薄壁硼硅玻璃（佳- FIL ® TW150 - 3; WPI的，萨拉索塔，佛罗里达州）移液器。河豚吸管电阻均在1.5-2.5MΩ范围; </li><li>准备FM1 -​​ 43 ®（N -（3 triethylammoniumpropyl）-4 - （4 - （dibutylamino）苯乙烯）吡啶溴化物，“特殊包装”; Invitrogen公司，卡尔斯巴德，加利福尼亚州）的解决方案。首先，加入160μL蒸馏水，一小瓶FM1 -​​ 43（100毫克）® 1毫米的股票。长达一个星期，这股可保持在4 ° C。然后，添加5μLFM1 - 43 ® 1毫升高K +林格+ 1MM Trolox的®。用铝箔保护解决方案，并保持在4 °，直到使用C; </li><li>打开明亮的场光显微镜和搜索完好双极细胞。稍微挖掘显微镜，以确保该神经元牢固地附着在箱体底部; </li><li>灌流笔感兴趣的细胞接近的位置，不断灌注与低Ca 2 +林格准备; </li><li>关闭室内灯光，并添加一个红色的长通滤波器（即RG630;肖特，德国）的光路，以最大限度地减少了FM染料激发; </li><li>填写FM染料溶液10μL装载吸管，安装在显微吸管和吸管上的准备没有超压降低，直到它是你要加载的双极细胞在同一焦平面。确保你至少有两个电极持有：一个用于调频染料不能用于膜片钳，否则它可能污染的细胞内的解决方案; </li><li>在约10微米的轴突终端的距离，位置河豚开放，转动灌流系统和粉扑吸管超压10秒染料溶液; </li><li>关闭超压，无需移动吸管，等待30秒; </li><li>转动灌流和沐浴室ADVASEP - 7的溶液中5分钟。在此期间，从浴缸中删除河豚吸管; </li><li> 5分钟后，切换到低Ca 2 +林格和灌注室25-30分钟，以便清除多余的染料。 </li></ol>第3部分：膜片钳和TIRFM成像<ol><li>虽然准备洗出，拉补丁的吸液管，厚壁硼硅玻璃（B150 - 86 - 10;萨特仪器公司，诺瓦托，CA）。补丁吸管电阻8-10MΩ范围; </li><li>洗出后，放置在视野的中心轴突的终端; </li><li>填充内部解决方案7μL的一个补丁吸管，挖掘移液器除去气泡，大衣与熔融的牙科用蜡（粘粘的蜡;克尔公司，橙，CA）的吸管，并安装在显微; </li><li>打开移液器的超压和吸管慢慢降低到准备。检查吸液管电阻在放大器和正确的吸管偏移，并与各自的放大器控制电容; </li><li>切换灌流控制振铃的。为了创建一个吸管和细胞之间gigaseal，稍微触摸电极对细胞体内的一角，打开吸管超压，一边轻轻地运用负压电极; </li><li>虽然密封，在放大器的选择“全细胞”模式，并设置单元格的控股潜力-60毫伏; </li><li>选择一个感兴趣的区域，涵盖整个轴突终端与成像软件，打开明亮的领域轻客，并简要地暴露（30毫秒）终端488 nm激光，找到合适的TIRF成像焦平面; </li><li>打破使用“ZAP”放大器的命令，而采用微负压吸管进入细胞; </li><li>纠正细胞电容和串联电阻，然后应用的利益的电压协议，同时成像的突触小泡的运动。正是在这个阶段，重要的是要有电压协议同步摄像头的帧速率。在我们的例子中，每帧30毫秒长，所以电压的变化发生在该值的倍数（即每300毫秒或10帧）; </li><li>试验之间至少等待40秒，以便恢复; </li><li>要检查突触丝带的位置，拍照，同时转动561 nm的激光。 </li></ol><img src="/files/ftp_upload/1305/fig1.jpg" alt="图1" /> 图1：实验装置。 488 nm激光（蓝色）的重点是向外围的客观焦平面和遭受内部全反射，当它到达的玻璃水介质界面。反射的激光束所产生的电磁场激发加载到最接近的博特的突触小泡的荧光OM的玻璃容器中，然后发出荧光（绿色）。荧光灯，然后引导到观察者的眼睛（描绘）或CCD相机。成像细胞的膜电位控制，同时通过补丁夹紧。这种方法允许传入的信号（膜电压）和神经元的输出（胞吐）之间的关系的研究。 <img src="/files/ftp_upload/1305/fig2.jpg" alt="图2" /> 图2：典型的结果。左：明场像一个孤立的金鱼双极细胞。右上：TIRF双极细胞的轴突终端突触囊泡形象加载带有FM 1-43 ®和488 nm激光（调频染料）成像。右下：修补细胞和轴突终端与561 nm激光成像后的同一个终端的形象。突触带标记的罗丹明基于肉眼结合肽（Rpep_rhod）。 表1：特定的试剂和仪器。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 名称</td><td>类型</td><td>制造商</td><td>目录</td><td>评论</td></tr><tr><td> - </td><td>空气球台</td><td> Newport公司</td><td> - </td><td> - </td></tr><tr><td> IX70 </td><td>倒置显微镜</td><td>奥林巴斯</td><td> - </td><td>配有一个钨灯亮场和横向开放港口为TIRF </td></tr><tr><td> TH4 - 100 </td><td>灯泡电源</td><td>奥林巴斯</td><td> - </td><td> - </td></tr><tr><td> FF498_581 - DI01 </td><td>分色过滤器</td><td> Semrock </td><td> - </td><td> - </td></tr><tr><td> NF01 - 405_488_568 </td><td>发射滤光片</td><td> Semrock </td><td> - </td><td> - </td></tr><tr><td>阿婆X100 Ø人力资源</td><td>目的</td><td>奥林巴斯</td><td> - </td><td>不适用1.65 </td></tr><tr><td> RG630 </td><td>红色玻璃过滤器</td><td>肖特</td><td> - </td><td> - </td></tr><tr><td> - </td><td> 488 nm激光</td><td>连贯</td><td> - </td><td>使用最小的功耗</td></tr><tr><td> - </td><td>快门</td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> VMM - D1 </td><td>快门驱动</td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> - </td><td> 561 nm激光</td><td>梅勒斯Griot </td><td> - </td><td> - </td></tr><tr><td> - </td><td>快门</td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> VMM - D3 </td><td>快门驱动</td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td>灌注压力套件</td><td>灌流系统</td><td>自动化科学</td><td> 09-04 </td><td> - </td></tr><tr><td>灌注笔</td><td>灌流系统</td><td>自动化科学</td><td> - </td><td> - </td></tr><tr><td> Valvelink 8 </td><td>灌流系统控制器</td><td>自动化科学</td><td> - </td><td> - </td></tr><tr><td>级联512B </td><td> EM CCD相机</td><td>罗珀科学</td><td> - </td><td> - </td></tr><tr><td> Metamorph 7.1 </td><td>图像处理软件</td><td>分子器件</td><td> - </td><td> - </td></tr><tr><td> EPC - 9 </td><td>膜片钳放大器</td><td> HEKA Elektronik公司</td><td> - </td><td> - </td></tr><tr><td>脉冲</td><td>放大器软件</td><td> HEKA Elektronik公司</td><td> - </td><td> - </td></tr><tr><td> MP - 285 </td><td>微操作机器人</td><td>萨特仪器</td><td> - </td><td> - </td></tr><tr><td> - </td><td>电极支架</td><td> HEKA Elektronik公司</td><td> - </td><td>外径为1.5毫米玻璃，2个单位</td></tr><tr><td>佳- FIL ® TW150 - 3 </td><td>高硼硅毛细管玻璃</td><td> WPI </td><td> - </td><td>没有灯丝</td></tr><tr><td> B150 - 86 - 10 </td><td>高硼硅毛细管玻璃</td><td>萨特仪器</td><td> - </td><td>随着长丝</td></tr><tr><td> P - 97 </td><td>微电极拖轮</td><td>萨特仪器</td><td> - </td><td>配备的3x3方块长丝和环境试验箱</td></tr><tr><td> - </td><td>压力真空气泵</td><td>托马斯科学</td><td> 7893B05 </td><td>创建真空去除液体的移液器商会和超压</td></tr><tr><td> MatLab的R2008a </td><td>分析软件</td><td> MathWorks公司</td><td> - </td><td> - </td></tr><tr><td> 353001 </td><td> 35 mm塑料培养皿</td><td>鹘</td><td> - </td><td> - </td></tr><tr><td> - </td><td>高折射率玻璃</td><td> PlanOptik </td><td> - </td><td>折射率为488 nm = 1.78 </td></tr><tr><td> M系列</td><td>高折射率液体</td><td> Cargille实验室</td><td> - </td><td>折射率= 1.78 </td></tr><tr><td> Sylgard 184 </td><td>硅弹性体套件</td><td>道康宁</td><td> - </td><td> - </td></tr><tr><td>谷胱甘肽</td><td>三肽</td><td>电解二氧化锰化学品</td><td></td><td>免费雷克升清道夫</td></tr><tr><td>透明质酸</td><td>酶</td><td>西格玛</td><td> H6254 </td><td> V型</td></tr><tr><td> L -半胱氨酸</td><td>氨基酸</td><td> Fluka公司</td><td> 30090 </td><td>激活木瓜</td></tr><tr><td>木瓜蛋白酶</td><td>酶</td><td> Fluka公司</td><td> 76220 </td><td>从番木瓜 </td></tr><tr><td> Trolox的® </td><td>可溶性维生素E </td><td>西格玛</td><td> 56510 </td><td>自由基清除剂</td></tr><tr><td> ADVASEP - 7 </td><td>磺化 β-环糊精衍生物</td><td>西格玛</td><td> A3723 </td><td>减少调频1-43 ®背景荧光</td></tr><tr><td> FM 1-43 ® </td><td>荧光染料</td><td> Invitrogen公司</td><td> T35356 </td><td> “特别包装” </td></tr><tr><td>粘粘的蜡</td><td>移液器涂料代理</td><td>科尔公司</td><td> - </td><td>减少移液器电容</td></tr></tbody></table> 表2：本研究采用的生理的解决方案。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 物质</td><td>低Ca 2 +林格</td><td>控制林格</td><td>高K +林格</td><td>内部解决方案</td></tr><tr><td>氯化钠</td><td> 120毫米</td><td> 120毫米</td><td> 97.5 mM的</td><td> - </td></tr><tr><td>氯化钾</td><td> 2.5毫米</td><td> 2.5毫米</td><td> 25毫米</td><td> - </td></tr><tr><td> MgCl 2的 </td><td> 1毫米</td><td> 1毫米</td><td> 1毫米</td><td> 4毫米</td></tr><tr><td> 氯化钙 </td><td> 0.5毫米</td><td> 2.5毫米</td><td> 2.5毫米</td><td> - </td></tr><tr><td> HEPES </td><td> 10毫米</td><td> 10毫米</td><td> 10毫米</td><td> 10毫米</td></tr><tr><td> EGTA </td><td> 0.75毫米</td><td> - </td><td> - </td><td> 0.5毫米</td></tr><tr><td>血糖</td><td> 10毫米</td><td> 10毫米</td><td> - </td><td> - </td></tr><tr><td>谷胱甘肽</td><td> 2毫米</td><td> 2毫米</td><td> - </td><td> 1毫米</td></tr><tr><td> CH 3 CSO 3 S * </td><td> - </td><td> - </td><td> - </td><td> 100毫米</td></tr><tr><td> TEACl </td><td> - </td><td> - </td><td> - </td><td> 10毫米</td></tr><tr><td> ATP镁</td><td> - </td><td> - </td><td> - </td><td> 10毫米</td></tr><tr><td> GTP李</td><td> - </td><td> - </td><td> - </td><td> 1毫米</td></tr><tr><td> RPEP - rhod ** </td><td> - </td><td> - </td><td> - </td><td> 5毫米</td></tr><tr><td>卷</td><td> 200毫升</td><td> 100毫升</td><td> 5微升</td><td> 100μL， </td></tr></tbody></table> *铯磺酸​​。 **肉眼结合肽：罗丹明+ EQTVPVDLSVARDR - COOH（兆瓦1997.75）。

<ol>
	<li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+microscopy+in+cell+biology.">Total internal reflection fluorescence microscopy in cell biology.</a> Traffic. 2, 764-774 (2001).</li><li>Zenisek, D., Steyer, J. A., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport,+capture+and+exocytosis+of+single+synaptic+vesicles+at+active+zones.">Transport, capture and exocytosis of single synaptic vesicles at active zones.</a> Nature. 406, 849-854 (2000).</li><li>Zenisek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+association+and+exocytosis+at+ribbon+and+extraribbon+sites+in+retinal+bipolar+cell+presynaptic+terminals.">Vesicle association and exocytosis at ribbon and extraribbon sites in retinal bipolar cell presynaptic terminals.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 4922-4927 (2008).</li><li>Zenisek, D., Horst, N. K., Merrifield, C., Sterling, P., Matthews, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+synaptic+ribbons+in+the+living+cell.">Visualizing synaptic ribbons in the living cell.</a> J. Neurosci. 24, 9752-9759 (2004).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li><li>Rouze, N. C., Schwartz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continous+and+transient+vesicle+cycling+at+a+ribbon+synapse.">Continous and transient vesicle cycling at a ribbon synapse.</a> J. Neurosci. 18, 8614-8624 (1998).</li></ol>

客观型TIRF显微镜的优点是：1）激发光在焦平面的客观限制在一个狭小的区域提供优良的光学切片，从而最大限度地减少了集中光线; 2）由于光线与距离成指数下降荧光强度的变化，可以在垂直方向的运动进行监测; 3）通过高数值孔径的目标1,5的高效率的光收集。 该技术的主要缺点是，它是有限的成像事件发生在100纳米的细胞表面，这是大致相当于一个在电子显微镜超薄切片。因此，坚持以玻璃的?...

imaging exocytosis in retinal bipolar cells with tirf microscopy

共有内部反射荧光（TIRF）显微镜是一种技术，使细胞膜发生的事件的研究是最接近高折射指数物质，如玻璃的荧光分子的选择性成像，<sup&gt; 1</sup&gt;。在这篇文章中，我们应用此技术从金鱼视网膜视网膜双极细胞的突触囊泡的胞吐形象。这些神经元是非常适合这种学习，由于其巨大的轴突终端。同时夹紧双极细胞的修补程序，它是可能的调查之间的突触前电压和突触释放的关系<sup&gt; 2,3</sup&gt;。突触小泡内的双极细胞端子装有一个荧光染料（调频1-43 ®）合作膨化染料和林格液含有高K<sup&gt; +</sup&gt;到突触终端的浓度。这种去极化的细胞，刺激成谷氨酸囊泡内吞作用和随之而来的上染率。洗涤多余的染料，大约30分钟后，细胞被夹住的补丁，并同时与488 nm激光成像准备。该补丁吸管解决方案包含一个基于罗丹明的肽结合选择性突触带状蛋白质肉眼<sup&gt; 4</sup&gt;，从而标签色带特别是当终端与561 nm激光成像。这使得活跃区的精确定位和分离的突触的突触事件。

Watch this Scientific Journal Video about Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy at JoVE.com

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

在这个视频中，我们演示了如何标记和可视化单突触囊泡的胞吐贩卖和使用总内部反射荧光（TIRF）显微镜在金鱼视网膜双极细胞。

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

imaging-exocytosis-in-retinal-bipolar-cells-with-tirf-microscopy

Research

JoVE Journal

Biology

11.6K 次观看.  Yale University School of Medicine. 在这个视频中，我们演示了如何标记和可视化单突触囊泡的胞吐贩卖和使用总内部反射荧光（TIRF）显微镜在金鱼视网膜双极细胞。

视频: Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

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 ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

成像 FITC-葡聚糖作为胞吐作用调节的记者

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...