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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucagon-like+peptide+1+promotes+satiety+and+suppresses+energy+intake+in+humans.">Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans.</a> The Journal of clinical investigation. 101, 515-520 (1998).</li><li>Hayworth, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+ATUM+ultrathin+section+libraries+with+WaferMapper:+a+multi-scale+approach+to+EM+reconstruction+of+neural+circuits.">Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits.</a> Frontiers in neural circuits. 8, 68 (2014).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI&#160; 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&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>14120</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid releasing agent&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>70880</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid silver colloidal&#160;&#160;&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>12630</td>
			<td></td>
		</tr>
		<tr>
			<td>CircuitWorks conductive epoxy&#160;&#160;</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&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma VP Scanning Electron Microscope&#160;</td>
			<td>Carl Zeiss&#160;</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 16GB of RAM or more</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data visualization software Imaris 7.5&#160;</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/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/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/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

Research

JoVE Journal

Biology

11.3K 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

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 ...

科研

生物学

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 ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

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 ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

使用新型机器人系统 Spotiton 进行时间解决研究的低温电子微观网格准备

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

通过 3D 相关聚焦离子束铣削制备样品，用于高分辨率冷冻电子断层扫描

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view.
In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification.
In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.

内源性LC3的超微结构定位-截面相关光电子显微镜

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal ...

使用 mtHyper7 对活酵母细胞中的线粒体过氧化物进行成像

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

作者聚焦：推进线粒体研究 - 用于亚细胞分析的 mtHyper7 生物传感器 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

mtHyPer7（一种用于线粒体过氧化物的比例生物传感器）在活酵母细胞中的成像

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...