Name: correlative confocal and 3d electron microscopy of a specific sensory cell
Uploaded: 2015-07-19T14:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell at JoVE.com

Our sincere appreciation is expressed to the following people: Drs. Sam Johnson and Benjamin Carlson of the Duke Light Microscopy Core Facility for their assistance with data visualization software, and Ms. Valerie Lapham and Dr. John M. Mackenzie, Jr. of the Center for Electron Microscopy at North Carolina State University for their advice on electron microscopy. We thank Dr. Elaine B. Boh&#243;rquez for her editorial assistance. Authors contributed in the following manner: DVB, SM, and RAL designed experiments and analyzed data. DVB performed experiments and FH performed manual rendering of data. SM is director of Renovo Neural, where SBEM data was acquired. DVB wrote the manuscript and all authors reviewed and edited the final manuscript. This work was supported by NIH grants R01DK091946 and Veterans Affairs grant I01BX002230 to RAL, and F32DK094704, to DVB.

所有的动物保健和实验，按照批准的杜克大学机构动物护理和使用委员会的协议进行。 Cocem3D从先前公布的协议10,11的组合和朵朵在这里用于研究使用PyyGFP记者12鼠标特定的肠内分泌细胞。使用市售的转基因报告小鼠这种方法可以容易地应用于其他感兴趣的细胞。相关共焦显微镜，串行块面扫描电子显微镜（SBEM），和数据呈现在3D:该方法在三个部分中描述。 相关共聚焦显微镜这一节的目的是收获自尺寸允许相关共焦和SBEM成像的远端结肠组织段。协议如下: 1.收获的组织<ol><li>准备将10ml PBS中50μg/ ml的肝素溶液。 </li><li>通过混合10毫升氯胺酮（100毫克/毫升）和1ml甲苯噻嗪（20毫克/毫升）制备甲苯噻嗪/氯胺酮麻醉的库存。 <ol><li>稀释甲苯噻嗪/氯胺酮股票1:4在0.1M PBS。 </li></ol></li><li>制备100毫升含有4％多聚甲醛和0.1％戊二醛的PBS中固定剂溶液。 </li><li>麻醉一个PyyGFP鼠标，6〜10周龄，用致死剂量的甲苯噻嗪/氯胺酮麻醉。腹腔注射麻醉剂以每20克鼠标重量0.15毫升</li><li>确认正确鼠标麻醉按捏的尾巴或脚趾，并使用软膏的眼睛，以防止干燥。 </li><li>使用一个可变流量的蠕动泵灌注固定液心内10。用手术剪（13厘米长）剖开腹腔，露出肠子，心脏和肺。保持心脏窄图案弯钳（12厘米长），并插入来自蠕动泵蝴蝶针（19克）我NTO左心室。随即，切右心房与弹簧剪刀直（长度为4毫米）。灌注以2ml / min的速率首先用1分钟肝素溶液，然后用15分钟冰冷固定液直到小鼠的尾巴是完全刚性的。 注意:灌注以缓慢的速度，以防止小血管破裂在肠粘膜是非常重要的。血管破裂会危及组织的超微结构。如果灌注量充足，肝脏应在3-5分钟变成苍白色。 </li><li>用小剪刀打开腹腔并切除从与盲肠到远端直肠交界整个结肠。放置在冰冷的PBS中的组织。虽然淹没在PBS，剖开小弹簧剪刀沿肠系膜结肠。 </li></ol> 2.解剖和修复组织片段<ol><li>使用手术刀，从远端结肠削减约6小组织片段（2平方毫米 ）。这样做在一张纸上Ø˚F牙模和淹没在几滴PBS的。 </li><li>在4℃下后固定在固定剂溶液中的组织片段3小时。 </li></ol> 3.微解剖组织块<ol><li>制备5％低熔点琼脂糖在PBS中并保持在水浴中于45℃。 </li><li>嵌入的组织片段在5％低熔点琼脂糖使用小塑料容器，如标准尺寸的组织-Tek的Cryomold。 </li><li>安装嵌入部分在振动刀切片机，并填写缓冲液盘与冰冷的PBS。 </li><li>削减300微米组织带0.8的幅度和0.04毫米/秒的速度。注意:在这一点上，将组织条将从琼脂糖抛下。 </li><li>重新嵌入300微米组织带在琼脂糖并安装它们切片垂直于vibratome的刀片。 </li><li>使用振动，切割组织带，其厚度为50微米。注意:最终组织块应为约300微米宽×50微米厚。这些ðimensions从导频共焦实验结果，表明肠内分泌细胞的厚度为15-20微米，neuropod的长度30-70纳米之间优化。因此，一个完整的细胞可以包含组织300微米宽的一个块内通过50微米厚的，如果细胞的取向是平行于组织块的面。变化这些尺寸取决于所感兴趣的组织和细胞类型。 </li><li>存储在PBS中的组织块，在4℃。 </li></ol> 4.共焦成像<ol><li> 4000:通过在1在PBS稀释的DAPI准备一个核染色解决方案。 </li><li>孵育在DAPI核染色的组织块5分钟。注意:DAPI染色通过细胞核，这是用于关联的共焦和SBEM图像有益便于区分单个细胞。 </li><li>在充电载玻片山组织块，加入几滴的PBS，并用盖玻片覆盖它们。 </li><li>利用共聚焦显微镜，确定块s的完整绒毛和感兴趣PyyGFP细胞和成像他们获得的z堆栈光学切面。 <ol><li>使用20X / 0.8蔡司计划复消色差透镜的目标，以获得Z-栈1微米的光学切片。 </li><li>用于Z-栈，使用的声道为405nm的吲哚（DAPI），以确定关系到其他细胞，488纳米内源性的GFP本地化感兴趣的细胞，和微分干涉对比（DIC），以确定到细胞相对的位置内腔。 </li><li>使用1,024像素或更高的图像分辨率。 </li></ol></li></ol> 5.嵌入块琼脂糖<ol><li>制备10ml含有4％多聚甲醛和2.5％戊二醛的PBS中固定剂。 </li><li>通过小心地加入的PBS在盖玻片的边缘滑动盖玻片远离载玻片而不损伤组织块中删除从载玻片上的组织块。然后，使用一个精美的艺术画笔从幻灯片转移的组织块到10.5毫升含有4％多聚甲醛和2.5％戊二醛固定剂的离心管。 </li><li>定位后的组织块O / N在4℃。 </li><li>转移的组织块PBS。然后，嵌入块平板在5％由两片玻璃载片之间夹入它们的低熔点琼脂糖。 注:嵌入在薄层琼脂糖块染色过程中简化后的操作。 </li><li>切琼脂糖在一个正方形，并​​作出缺口上风侧。注意:该步骤是必要的，以保持在后续步骤中的取向。 </li><li>商店块在1.5 ml离心管，用PBS于4℃直至进一步处理。 </li></ol>串行块面部扫描电子显微镜（SBEM） 在本节中，将组织块制备和成像的SBEM在低倍率。块表面的观察图像，然后用共焦数据相关，以确定含有目的细胞的区域。一旦该区域被识别，所述组织被成像在为7nm /像素和为70nm的切片的分辨率。这是足以解决和其他细胞器区分大致密核心分泌囊泡。在肠内分泌细胞中，这种类型的小泡的100至150纳米，直径13变化。协议如下: 6.染色组织切片<ol><li>从PBS中去除组织和冲洗在0.1M二甲胂酸盐缓冲液三次，每次5分钟，。 </li><li>溶解在0.1M二甲胂酸盐缓冲液染色块1小时在0.1％的单宁酸，以提高细胞膜14的对比度。 </li><li>根据公布的协议11进行随后的染色和组织的脱水。以下Deerinck等。协议，准备这些解决方案:1）01％（重量/体积）thiocarbonhydrazide（THC）; 2）导致天冬氨酸溶液; 3）1％乙酸双氧铀，和4）锇tetraoxyde /亚铁氰化钾（OSO 4 / K亚铁氰化物）。混合4％OSO 4倍和2倍ķ亚铁股票以10.3克ķ亚铁和0.86克娜卡可酸盐在10毫升H 2 O]:1的比例，使OSO 4 / K亚铁。注:具有嵌入在琼脂糖组织染色过程中便于操作。小心取出琼脂糖后续树脂浸润。 </li><li>冲洗样品三次，每次5分钟，在0.1M二甲胂酸盐缓冲液，然后染色用的OsO 4 / K氰化钾溶液2小时，在4℃。 </li><li>冲洗在超纯水中三次，每次5分钟，并与TCH染色30分钟，在60℃。 </li><li>冲洗在超纯水中三次，每次5分钟，并染色以在室温60分钟2％OSO 4（不需K亚铁氰化物）。在超纯水中再漂洗三次，每次5分钟。 </li><li>染色用1％醋酸铀O / N在4℃。冲洗在超纯水中三次，每次5分钟。 </li><li>与染色在60℃铅天冬氨酸30分钟。冲洗在超纯水中三次，每次5分钟。 </li><li>通过在漂洗脱水搜索解决方案组织纳秒与增加的乙醇浓度。冲洗2次，每次5分钟，用50％，75％，85％，95％，和绝对的100％乙醇。在结束时，冲洗部分与环氧丙烷2次，每次10分钟。 </li></ol>在树脂7浸润和嵌入组织切片<ol><li>浸润块使用市售avaialble套件树脂。采用EPON嵌入工具包中的树脂嵌入组织。制备48％环氧，20％毫升DDSA，30％毫升NMA，和2％DMP30的树脂混合。搅动树脂混合物剧烈5分钟，然后将混合物在真空中进行30分钟，以允许气泡表面化。 </li><li>混合树脂，环氧丙烷在1:1的比例，大力摇晃。从组织中移除最终脱水漂洗，并添加树脂与环氧丙烷混合。地方小瓶在转子Ø采样/ N和离开瓶不封顶。第二天，添加新鲜的EPON树脂和混合90分钟在旋转器。 </li><li>嵌入块在树脂尽可能平坦夹着它们之间载玻片已固化液体分离剂，以防止它们粘在一起15。 </li><li>一旦块被嵌入平面，固化该块的加成48小时，在60℃。 </li><li>拉滑动开以释放块。 </li></ol> 8.安装和修整成像的组织块<ol><li>下解剖范围，匹配嵌入树脂的共焦显微照片，以方便识别修剪块之前包含感兴趣的细胞的区域中的组织块的方向。 </li><li>手动降到〜500×500微米的块面修剪树脂包埋块。 </li><li>安装块平放在含有导电环氧树脂干30分钟的脚。然后，躺在表面干燥O / N块持平，为60℃。 </li><li>大衣胶体银液块。保持组织切片平，以确保该块的切片以直角以促进共rrelating串行块面和共聚焦显微照片。 </li></ol> 9. SBEM <ol><li>图像使用扫描电子显微镜配备有SBEM系统的块（例如.G。，3查看）。 </li><li>在〜2微米为单位设置的初始厚度部分，切，直到组织的脸上浮现从块。 </li><li>获得整个块面的surver图像。 </li><li>斐济使用软件，无论采取串行块面和共聚焦显微测量过程中固定和染色16来生成一个共同点，占样本翘曲。 </li><li>由分母乘以在共焦图象的坐标定位对块面感兴趣的区域。还可以使用其他的结构特征，如微绒毛，杯状细胞，或固有层的位置，作为参考。 </li><li>感兴趣的图像区域中的2.25千伏，在高真空模式为7nm /像素（或15,147X放大率）。切片增量应设置在70纳米或更低。 </li><li>收集原始16位.dm3格式SBEM数据。 </li></ol>对于曲面细分10 SBEM优化图像<ol><li>从原材料.dm3格式SBEM图像转换为8位.TIFF。 </li><li>过滤器使用.TIFF在斐济一个0.8高斯模糊滤镜图像。 </li><li>分频设置为原始大小的25％的数据，并将其保存为.TIFF栈，以尽量减少需要处理的一套RAM的内存量。 </li><li>使用对齐斐济插件"与SIFT线性栈对齐"的翻译模式SBEM图像的堆栈，并使用"裁剪3D"插件裁剪。注:裁切有助于进一步减少RAM存储器的必需的量用于分割和体绘制。 </li></ol>在3D渲染数据 11.共聚焦显微镜<ol><li>重建使用自动超越了"面"工具的模式Z-栈。 图1D，显示了一个REC使用上，该表面区域细节的平滑选项的每个信道的。施工在0.126微米，和阈值作为绝对强度。 </li></ol> 12.串行块面部扫描电镜注意:手动数据分割是耗时的过程并且根据要呈现的过程可能需要几个星期的特征的大量时间。分割为Bohórquez 等人提出的视频和数字2014 8手工完成，花了大约500小时的劳动。建议优先的基本特征开始分割处理之前需要渲染。的步骤如下: <ol><li>用"表面"工具在绘制模式和"轮廓"选项在绘制的50毫秒模式手动段和体绘制的感兴趣的细胞。 </li><li>追踪细胞的每一片流畅的渲染或每5片更快的渲染上的轮廓。 </li><li>出口最终IM使用"快照"工具在300 dpi或以上的分辨率青睐。 </li></ol>

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萨蒂什Medicetty博士是员工和Renovo公司神经公司获得100％的工资;然而，这并不改变笔者的坚持朱庇特的政策对共享数据和资料。没有利益冲突的声明剩余的作者。

<html>胃肠chemosensation正在成为生物医学研究中的一个令人兴奋的新领域。这在很大程度上是由于功能性味觉受体在肠内分泌细胞18的发现。随后的研究已经表明，肠内分泌细胞表达特异受体为营养物质，包括碳水化合物，脂质和氨基酸5,6,19,20。对于这些发现的催化因素是报告小鼠的发展，其中，肠内分泌细胞被荧光标记的20。我们开发了这些小鼠中，PyyGFP鼠标，来研究远端小肠和结肠17,21的肠内分泌细胞。这些细胞是感兴趣的，因为它们分泌PYY和胰高血糖素样肽1，这两者都是饱22,23的诱导剂。当时，这些细胞的完整超结构帐户是缺少，我们认为这是必要的，了解他们的信令机制。 内容】"&gt;在这里，我们描述了一种在视觉上的方法，以消除共聚焦显微镜用SBEM。该方法被称为Cocem3D，这使我们能够记录肠内分泌细胞的完整超微结构。我们已经报道，这些细胞具有包含三一个neuropod四分之三的所有分泌囊泡8组成。内neuropod，有神经丝和很像神经轴突，neuropods由肠溶胶质细胞8培育出来的。更重要的是，它是尽管这些neuropods该肠内分泌细胞物理连接到神经元支配肠和结肠9。 其中cocem3D的优势是它的简单。减小组织样本成可以通过共聚焦成像和SBEM便于组织内的特定细胞的识别的块。分泌囊泡和其它细胞器可以识别容易在为7nm /像素的分辨率。因为在SBEM的部分都将被丢弃，进一步PROCES唱组织，以确定特定的蛋白质是不在这一点上的选项。然而，方法如ATUM 24，其中来自组织块部分被保留的发展，很可能使特定的蛋白质在细胞的识别。对肠内分泌细胞确定化学感受受体的具体位置是用于药物治疗肥胖的发展至关重要的信息，因为肠内分泌细胞是在大脑中的肠道和饱足感的食物之间的感官接口。

生活在细胞内发生的时间和空间。随着时间的推移改变使用时间推移显微镜结合荧光成像技术，如超高分辨率显微镜经常研究。空间，特别是细胞或细胞 - 细胞相互作用内细胞器的结构中，只能由一个完整的帐户细胞的微细结构的衍生。综合考虑一个细胞的微细结构也能带来清晰基因组功能的情况下的基因组是可用的，像C.线虫线虫1或平面placozoa tricoplax粘着2。连续切片电子显微镜现在是一个可再现的，时间效率，和更便宜的任务由于自动化3D电子显微镜技术的发展，例如串行块面扫描电子显微镜3（SBEM）。 需要进行结构信息阐明的功能是在一定的CEL非常明显升类型，其中功能取决于物理的细胞 - 细胞相互作用，如神经元，神经胶质细胞，或感觉上皮细胞。我们是在阐明从在肠道的管腔养分感觉信号如何被转导到最终调制欲求行为的电信号特别感兴趣。该电路是复杂的，但开始在肠的壁，其中营养物质接触到感觉上皮细胞，称为肠内分泌细胞接触。不像其他感官上皮细胞，如味觉细胞，肠内分泌细胞在整个肠上皮的一个的比率分散到千4-7。因此，他们已经很难确定和研究，并且很长一段时间，他们只能作为胃肠激素的来源。但与细胞特异性的荧光报告小鼠的发展，这些细胞的复杂感觉功能正在出现。使用一个这些记者老鼠，肽YY-GFP（PyyGFP）小鼠，我们发现，enteroendocrINE细胞有显着的胞质尾，我们命名为neuropod。 neuropods的外观建议在细胞 - 细胞通信的保守功能。因此，我们推断，通过记录一个肠内分泌细胞的超微结构，neuropods的功能可以被衍生。 理解在分散细胞，其是难以识别的附属物的结构的需要是开发一种方法，以共聚焦显微镜和SBEM结合的主要理由。鉴定使用PyyGFP肠内分泌细胞特异报告小鼠感兴趣的细胞。该方法使我们能够记录的肠内分泌细胞及其neuropod整个超微结构。在neuropods，我们发现神经元轴突的结构特点，并neuropods外，我们发现肠神经胶质8肉体关系。的确，neuropods含有约70％的分泌囊泡表明在这些细胞的分泌功能中起重要作用的。建筑上的结构数据，最近我们发现，通过这些neuropods，肠内分泌细胞和神经元支配肠道形成神经上皮电路，在舌头8,9类似口味细胞。 使用此相关显微镜方法揭开的化学感受胃肠等机制的特点，从结构数据收集源于。我们认为这种方法可以是在细胞生物学的其他领域，特别是在细胞组织内以非常低的比率分散有用。我们提到的方法，相关的共聚焦和串行块面部扫描电子显微镜在三维（Cocem3D）。该方法包括以下主要步骤:组织解剖，共焦显微镜，SBEM成像，SBEM和共焦图象的相关性，并手工分割。相对于其他相关方法，这个概念是相当简单的，因为相关的物理而不是化学。

<table><tbody><tr><td>Phosphate buffered saline</td><td>Life technologies</td><td>10010023</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H4784</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>158127</td><td>4%, freshly made in PBS, final pH 7.4</td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>G5882</td><td></td></tr><tr><td>Dental wax</td><td>Electron Microscopy Sciences</td><td>72660</td><td></td></tr><tr><td>Low-melting agarose</td><td>Life technologies</td><td>16520-100</td><td>5%, freshly made in PBS</td></tr><tr><td>Standard Tissue-Tek Cryomold</td><td>Electron Microscopy Sciences</td><td>62534-25</td><td></td></tr><tr><td>DAPI&amp;nbsp; nuclear stain</td><td>Life technologies</td><td>D1306</td><td></td></tr><tr><td>Postively charged glass slides and coverslips</td><td></td><td></td><td></td></tr><tr><td>Fine art paintbrush #1</td><td></td><td></td><td></td></tr><tr><td>Cacodylate buffer</td><td>Electron Microscopy Sciences</td><td>11650</td><td></td></tr><tr><td>Tannic acid</td><td>Electron Microscopy Sciences</td><td>21700</td><td></td></tr><tr><td>EMbed 812 kit&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>14120</td><td></td></tr><tr><td>Liquid releasing agent&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>70880</td><td></td></tr><tr><td>Liquid silver colloidal&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>12630</td><td></td></tr><tr><td>CircuitWorks conductive epoxy&amp;nbsp;&amp;nbsp;</td><td>ITW Chemtronics</td><td>CW2400</td><td></td></tr><tr><td>Variable flow peristaltic pump</td><td>VWR</td><td>70730-064</td><td></td></tr><tr><td>VT1200S Vibrating blade microtome</td><td>Leica</td><td></td><td></td></tr><tr><td>Zeiss 780i confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td></td><td></td></tr><tr><td>Sigma VP Scanning Electron Microscope&amp;nbsp;</td><td>Carl Zeiss&amp;nbsp;</td><td></td><td></td></tr><tr><td>3view system</td><td>Gatan</td><td></td><td></td></tr><tr><td>Renovo Neural Inc (Cleveland, OH)</td><td></td><td>http://www.renovoneural.com</td><td>Renovo provides 3d EM services</td></tr><tr><td>Fiji software</td><td>Open access software</td><td></td><td></td></tr><tr><td>Computer station with 16 GB of RAM or more</td><td></td><td></td><td></td></tr><tr><td>Data visualization software Imaris 7.5&amp;nbsp;</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

correlative confocal and 3d electron microscopy of a specific sensory cell

一个细胞的超微结构的划分对于理解它的功能很重要。这可以是在整个制成多种类型的细胞，如肠上皮的内分泌细胞的组织扩散稀有细胞类型是一项艰巨的项目。食物和细菌的这些胃肠传感器已很难研究，因为它们以1:1的比例分散在其他的上皮细胞:1,000。最近，转基因报告小鼠已经产生荧光的手段，以确定肠内分泌细胞。其中之一是将肽YY-GFP小鼠。使用这款鼠标，我们开发了一种方法来关联焦和串行块面部扫描电子显微镜。我们命名方法cocem3D并应用它来识别特定的肠内分泌细胞在组织中推出3D细胞的超微结构。 cocem3D的分辨率是足够的，以确定细胞器一样小分泌囊泡和区分细胞膜为卷重ndering。 Cocem3D可以很容易地适应研究其它特定的细胞类型的三维超微结构其天然组织中。

这里介绍的方法被用于研究特定细胞的超微结构的层上皮的范围内。在这种情况下，感兴趣的细胞是难以捉摸的肠内分泌细胞。其原位鉴定有可能仅在过去的几年中与细胞特异性荧光报告小鼠的发展。在2011年，我们开发了一种鼠标，其中激素PYY的启动子驱动的绿色荧光蛋白17的表达。在此PyyGFP鼠标，小肠和结肠的远侧部分的肠内分泌细胞在UV光下很容易区分。这种小鼠模型是一个基础，以确定含有肠内分泌细胞的组织块，并对其进行处理的SBEM。相关的方法在这里称为cocem3D，并建立在先前公布的协议10,11。 优化组织块的尺寸是用于相关的关键。在初步实验中，它被确定在组织块300微米长×50μm厚SBEM图像的数目被减少到最低限度，同时覆盖所关注的细胞的整个身体。 图1A示出了夹层的俯视使用振动叶片microtrome到获得与权利尺寸的组织块。至少有100块是通过它们的排序挑那些感兴趣的完整细胞之前获得。所选块通过共聚焦显微镜和图像的z栈成像光学地间隔开，每1微米（ 图1B和C）。图1D示出了肠内分泌细胞在小鼠的回肠体积渲染视图。其突出neuropod延伸上皮细胞下为60〜微米。 感兴趣的细胞是通过关联的共焦的z栈与整个块面的SBEM图像发现。一个例子是从PyyGFP的远端结肠这里提出一个块的鼠标。获得共焦的z栈（ 图2A）以后，该块被嵌入到一个薄层低熔点琼脂糖（ 图2B）。这是必要的后续处理的块。它保持块尽可能平坦，以匹配该光学片与SBEM切片的取向是很重要的。在图2C中 ，一个代表性的图像示出了组织块的整个面的。以前发表的该图的部分被在Bohorquez 等人，2014年8，这随后通过考虑组织的地标和尺寸相关的共聚焦图像。 图2D示出了该块的共焦和SBEM图像的重合，揭示了我们感兴趣的细胞的精确位置。 一旦细胞已被确定，SBEM成像完成时的分辨率足够高，以确定分泌囊泡和其他细胞器。该数据集介绍他重新进行成像以每像素为7nm作为该块的拍摄图像每70纳米。设置数据包含跨越45微米的组织深度643的图像。几微米的在整个块面的低倍率的初始成像剃光。原始SBEM数据集是在.dm3格式采集并转化为.TIFF进行后续处理。使用软件斐济插件"作物（3D）"块包含目的只有该地区SBEM堆栈被裁剪。这减少了所需要的体绘制的RAM存储器的量。该电池是由它的位置和分泌泡鉴定，并采用了Imaris软件分割。 图3包含了肠内分泌细胞的3D渲染。 <img alt="图1" src="/files/ftp_upload/52918/52918fig1.jpg" /> 图1:相关激光共聚焦显微镜（A）工作流程剖析了一块肠组织成一个组织块300×50微米厚。 （ 二）正确尺寸的代表组织块。需要注意的是从小鼠小肠这些尺寸的组织块跨越约3绒毛或，在结肠约8隐窝的情况。在包含感兴趣的细胞小肠（C）一个绒毛。 （D）的共焦Z堆叠使用了Imaris软件在3D渲染显示具有突出neuropod运行肠上皮下方的肠内分泌细胞。蓝= DAPI核染色。酒吧= 10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52918/52918fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52918/52918fig2.jpg" /> 图2:串行块面部扫描电子显微镜（A）一塞莱的共聚焦图像。从含有目的PyyGFP肠内分泌细胞（绿色）的结肠反恐执行组织块。 （B）中一旦块被共焦显微镜成像，它随后将嵌入平板在低熔点琼脂糖用玻璃载片。嵌入在琼脂糖组织块便于后续处理，并且在左上角的切口有助于装入SBEM内的块中的右方向。 （C）的 SBEM图像块的脸部。与焦SBEM数据（D）的相关性来确定利率（白色箭头）的细胞。在c和d该图的图像从Bohorquez 等改性，2014 8。蓝色= DAPI核染色。酒吧= 10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52918/52918fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52918/52918fig3.jpg" /> 图3:使用了Imaris软件肠内分泌细胞的数据呈现在3D（A）体积渲染。该单元格中包含挤满了分泌囊泡neuropod。对于肠内分泌细胞超微结构的完整描述，请参阅Bohórquez 等人 。2014年8。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52918/52918fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell at JoVE.com

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy.

一个具体的感觉细胞的共聚焦相关和3D电子显微镜

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

correlative-confocal-3d-electron-microscopy-specific-sensory

Universidad San Sebastian

Research

JoVE Journal

Biology

11.4K 次观看.  Duke University Medical Center. Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy. 

视频: Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

科研

生物工程

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

发育生物学

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

神经科学

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

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

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

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

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

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

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

生物学

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

免疫与感染

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201 
2 Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Confocal fluorescence microscopy is an imaging technique that enables increased optical resolution as compared to conventional &#34;wide-field&#34; epifluorescence microscopy. Confocal microscopes are able to achieve improved x-y optical resolution through &#34;laser scanning&#34;- typically a set of voltage-controlled mirrors (galvanometer or &#34;galvo&#34; mirrors) that direct laser-illumination to each pixel of the specimen at a time. More importantly, confocal microscopes achieve superior z-axial resolution by using a pinhole to remove out of focus light originating from locations that are not in the z-plane being scanned, thus enabling the detector to collect data from a specified z-plane. Because of the high z-resolution achievable in confocal microscopy, it is possible to collect images from a series of z-planes (also-called z-stack) and construct a 3D image through software.
Before discussing the mechanism of a confocal microscope, it is important to consider how a sample interacts with light. Light is composed of photons, packets of electromagnetic energy. A photon impinging on a biological sample can interact with the molecules comprising the sample in one of four ways: 1) the photon does not interact and passes through the sample; 2) the photon is reflected/scattered; 3) the photon is absorbed by a molecule and the absorbed energy is released as heat through processes collectively known as nonradiative decay; and 4) the photon is absorbed and the energy is then rapidly reemitted as a secondary photon through the process known as fluorescence. A molecule whose structure permits fluorescence emission is called a fluorophore. Most biological samples contain negligible endogenous fluorophores; therefore exogenous fluorophores must be used to highlight features of interest in the sample. During fluorescence microscopy, the sample is illuminated with light of the appropriate wavelength for absorption by the fluorophore. Upon absorbing a photon, a fluorophore is said to be &#34;excited&#34; and the process of absorption is referred to as &#34;excitation&#34;. When a fluorophore gives up energy in the form of a photon, the process is known as &#34;emission&#34;, and the emitted photon is called fluorescence.
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a &#34;focal point&#34; where it is maximally focused. Beyond the focal point the light again diverges. The entering and exiting beams may be visualized as a pair of cones touching at the focal point (see Figure 1, left panel). The phenomenon of diffraction imposes a limit on how tightly a beam of light can be focused - the beam actually focuses to a spot of finite size. Two factors determine the size of the focal spot: 1) the wavelength of the light, and 2) the light-gathering ability of the objective lens, which is characterized by its numerical aperture (NA). The focal &#34;spot&#34; extends not only in the x-y plane, but also in the z direction, and is in reality a focal volume. The dimensions of this focal volume define the maximum resolution achievable by optical imaging. Although the number of photons is greatest within the focal volume, the conical light paths above and below the focus also contain a lower density of photons. Any fluorophore in the light path can thus be excited. In conventional (wide-field) epifluorescence microscopy, emission from fluorophores above and below the focal plane contribute out-of-focus fluorescence (a &#34;hazy background&#34;), which reduces image resolution and contrast, as demonstrated in Figure 1, with the red cube representing fluorophore emission above the focal plane (red star) that results in out-of-focus fluorescence (top right). This problem is ameliorated in confocal microscopy, due to the utilization of a pinhole. (Figure 2, bottom right). As depicted in Figure 3, the pinhole allows emissions originating from the focal place to reach the detector (left), while blocking the out-of-focus fluorescence (right) from reaching the detector, thus improving both resolution and contrast.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig1.jpg" /> 
Figure 1. Optical resolution of epifluorescence versus confocal microscopy. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/10501/10501fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a focal volume and then diverges (left). The red star represents the focal plane of a sample that is being imaged while the red square represents fluorophore emission above the focal plane. When capturing an image of this sample using an epifluorescent microscope, the emission from the out-of-focus red square will visible and contribute to a &#34;hazy background&#34; (top right). Confocal microscopes have a pinhole which prevents the detection of light emitted outside of the focal plane, eliminating the &#34;hazy background&#34; (bottom right).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig2.jpg" /> 
Figure 2. Pinhole effect in confocal microscopy. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/10501/10501fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the highest intensity of the excitation light is at the focal point of the lens (left, red oval), other parts of the sample not in the focal point (right, red star) will get light and fluoresce. To prevent light emitted from these out-of-focus regions to reach the detector, a screen with a pinhole is present in front of the detector. Only the in-focus light (left) emitting from the focal plane is able to travel through the pinhole and reach the detector. The out-of-focus light (right) is blocked with the pinhole and fails to reach the detector.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig3.jpg" /> 
Figure 3. Principal components of a confocal laser scanning microscope. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/10501/10501fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For the sake of simplicity, the mechanistic description of a confocal microscope will be limited to that of the Nikon Eclipse Ti A1R. Although there may be minor technical differences between different confocal microscopes, the A1R serves well as a good model for describing confocal microscope function. The excitation light beam, produced by an array of diode lasers, is reflected by the primary dichroic mirror into the objective, which focuses the light onto the specimen being imaged. The primary dichroic mirror selectively reflects the excitation light while allowing light at other wavelengths to pass through. The light then encounters the scanning mirrors that sweep the light beam across the specimen in an x-y manner, illuminating a single (x,y) pixel at a time. Fluorescence emitted by fluorophores at the illuminated pixel is collected by the objective lens and passes through the primary dichroic mirror to reach an array of photomultiplier tubes (PMTs). Secondary dichroic mirrors direct the emission light to the appropriate PMT. Excitation light scattered by the sample back into the objective is reflected by the primary dichroic mirror back towards the specimen, and thus prevented from entering the detection light path and reaching the PMTs (see Figure 3). This allows the relatively feeble fluorescence to be quantified without contamination by light scattered from the excitation light beam, which is typically orders of magnitude more intense than the fluorescence. Because the pinhole blocks light from outside of the focal volume, the light arriving at the detector comes from a narrow, selected z-plane. Therefore, images can be collected from a series of adjacent z-planes; this series of images is often referred to as a &#39;z-stack&#39;. By using the appropriate software, a z-stack can be processed to generate a 3D image of the specimen. A particular advantage of confocal microscopy is the ability to distinguish the subcellular localization of staining. For example, the differentiation between membrane staining from intracellular staining, which is very challenging with conventional epifluorescence microscopy (1, 2, 3).
Sample preparation is an important facet of confocal imaging. A strength of optical microscopy techniques is the flexibility to image live or fixed cells. When attempting to produce 3D images, because of the number of images that must be acquired for a z-stack, the difficulty of maintaining cell health, and the movement of live cells and their organelles, the use of fixed cells is typical. The procedure for fixing and staining cells for confocal fluorescence is similar to that conventionally used in immunofluorescence. After culture in chamber slides or on coverslips, cells are fixed using paraformaldehyde to preserve cellular morphology. Non-specific antibody binding is blocked using bovine serum albumin, milk, or normal serum. In order to maintain the specificity of the secondary antibodies, the solution used should not originate from the same species in which the primary antibodies were generated. The cells are incubated with primary antibodies that bind the antigen of interest. When labeling several cellular targets, the primary antibodies must each be derived from a different species. Antibodies tagging an antigen are then bound by fluorophore-conjugated secondary antibodies. Fluorophore-conjugated secondary antibodies should be selected so that they are compatible with the wavelengths of laser excitation available in the confocal microscope. When visualizing multiple antigens, the excitation/emission spectra of the fluorophores should differ enough so that their signals can be discriminated by microscopic analysis. The stained specimen is then mounted on a slide for imaging. A mounting medium is used to prevent photobleaching and specimen dehydration. If desired, a mounting medium containing a nuclear counterstain (e.g. DAPI or Hoechst) can be used (4).
In the following protocol, mouse fibroblasts transfected to express CD1d (LCD1) were stained with antibodies recognizing CD1d and CD107a (LAMP-1). CD1d is a major histocompatibility complex 1 (MHC 1)-like receptor present on the surface of antigen presenting cells that presents lipid antigens. LAMP-1 (lysosomal associated membrane protein-1) is a transmembrane protein primarily present in lysosomal membranes. For proper antigen presentation, CD1d is trafficked through the low pH lysosomal compartment, so LAMP-1 is being used as a marker of the lysosomal compartment for this protocol. By probing the LCD1 cells with anti-CD1d and anti- LAMP-1 that were produced in different species, secondary antibodies with unique fluorophores can be used to determine the localization of each protein in the cell and whether CD1d is present in the LAMP-1 positive lysosomal compartments.

Confocal Fluorescence Microscopy and Localization of Proteins

共聚焦荧光显微镜:确定小鼠纤维细胞中蛋白质定位的技术

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine ...

一个具体的感觉细胞的共聚焦相关和3D电子显微镜

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相关显微镜的动力相互作用的三维结构分析