该项目得到了国家自然科学基金（32201235傅志飞）、福建省自然科学基金（2022J01287致傅志飞）、福建医科大学先进人才研究基金（XRCZX2021013傅志飞）、福建省金融专项科学基金（22SCZZX002致傅志飞）的支持。 国家卫健委非人灵长类动物生育力调控技术评价重点实验室、福建省妇幼保健院成立（2022-NHP-04致傅志飞）。我们感谢福建医科大学公共技术服务中心的Linying 周、Minxia Wu、习 Lin和Yan 胡在EM样品制备和EM成像方面的支持。

畜牧业和实验经福建医科大学医学中心机构动物护理与使用委员会批准。当前协议的分步工作流程如 图 1 所示。
1. 样品制备
<ol><li>小鼠大脑<ol><li>购买转基因小鼠（见 材料表）和寡核苷酸引物（见 材料表）对这些小鼠进行基因分型。</li>
		<li>按照先前发布的方案12,13 灌注并从颅穹窿中取出完整的大脑。</li>
		<li>用10mL固定溶液（参见 材料表）在4°C过夜。</li>
		<li>用0.1M磷酸盐缓冲液（PB）冲洗掉固定溶液3×10分钟。</li>
		<li>使用振荡切片机将小鼠大脑切成500μm厚度的切片（参见 材料表）。</li>
		<li>使用立体显微镜用手术刀将荧光区域切成小块（参见 材料表）。 注意： 小块的体积不得大于 1 mm3。</li>
	</ol></li>
	<li>培养细胞<ol><li>将 HeLa 细胞接种到 6 cm 细胞培养皿中，并将它们维持在生长培养基（Dulbecco 改良的 Eagle 培养基 [DMEM] 低葡萄糖，补充有 10% 胎牛血清 [FBS]）。</li>
		<li>第二天，按照转染试剂盒的说明书，使用DNA转染试剂（参见 材料表）将含有mScarlet的质粒转染到细胞中。</li>
		<li>转染后 48 小时，将转染的细胞胰蛋白酶消化并离心以获得 1,000 × g 的细胞沉淀。细胞沉淀称为 样品块。</li>
	</ol></li>
</ol>2. EM样品制备
<ol><li>固定<ol><li>使用固定溶液（参见 材料表）在4°C下固定样品块过夜。</li>
		<li>取出固定溶液，并用0.1M PB从样品块冲洗掉固定溶液3×10分钟。</li>
	</ol></li>
	<li>四氧化锇（OsO4）染色<ol><li>用OsO4 溶液（参见 材料表）在4°C下固定样品块1小时。</li>
		<li>取出OsO4溶液，并用ddH2O从样品块中冲洗掉OsO4溶液3×10分钟。</li>
	</ol></li>
	<li>醋酸铀酰（UA）染色<ol><li>使用2%UA溶液（参见 材料表）在4°C下对样品块进行染色1小时。</li>
		<li>取出UA溶液，用ddH2O冲洗样品块3×10分钟。</li>
	</ol></li>
	<li>梯度脱水<ol><li>用梯度乙醇脱水样品块，如下所示：50%10分钟，70%10分钟，80%10分钟，90%10分钟，2×10分钟在100%乙醇中。</li>
		<li>用无水丙酮将样品块脱水3×10分钟。</li>
	</ol></li>
	<li>树脂渗透<ol><li>用梯度树脂浸润样品块（见 材料表）：丙酮与树脂3：11 1小时，丙酮与树脂1：1 2小时，丙酮与树脂1：3 3小时。</li>
		<li>用纯树脂代替3×12小时。</li>
	</ol></li>
	<li>树脂包埋<ol><li>使用牙签将样品块转移到胶囊中（参见 材料表）。</li>
		<li>将纯树脂加入含有样品块的胶囊中，并在60°C的烘箱中聚合树脂14-16小时。</li>
	</ol></li>
</ol>3. 用金纳米颗粒涂覆盖板玻璃
<ol><li>盖玻片清洁<ol><li>用清洁缓冲液（参见 材料表）在142°C下洗涤盖玻片2小时。</li>
		<li>用ddH2O冲洗清洁缓冲液3×10分钟。</li>
		<li>小心地将盖玻片转移到装有无水乙醇的烧杯中并孵育过夜。</li>
		<li>在干净的工作台上将盖玻片风干几分钟。</li>
	</ol></li>
	<li>在盖玻片上涂上金纳米颗粒。<ol><li>使用玻璃水泥将载玻片固定在台式离心机旋转的中心（参见 材料表）。</li>
		<li>使用便利贴在载玻片的中央安装盖玻片。</li>
		<li>在盖玻片上加入 75 μL 的 1% pioloform（参见 材料表）。 注意：我们还测试了 Formvar（参见 材料表），它运行良好。</li>
		<li>在1,360× g 下旋转3分钟（图2A），以在盖玻片表面产生一层薄薄的pioloform。</li>
		<li>在室温下用250μL0.1%聚-L-赖氨酸（参见 材料表）孵育盖板表面1小时（图2B）。</li>
		<li>用ddH2O冲洗，然后将盖玻片风干几分钟。</li>
		<li>用ddH2O稀释80nm金纳米颗粒（见材料表）至25%（v / v，OD600 = 0.07）（见材料表）并超声处理15分钟。</li>
		<li>在室温下将盖玻片表面与稀释的金纳米颗粒孵育1小时（图2C）。</li>
		<li>用ddH2O冲洗，然后将盖玻片风干几分钟。 注意：在涂有 pioloform 和金纳米颗粒 2 周后，它效果很好。但我们建议在涂覆后立即使用。</li>
	</ol></li>
</ol>4.超薄切片
<ol><li>将样品块的表面修剪成直角梯形。使用金刚石刀（见 材料表）和超薄切片机（见 材料表）切割厚度为100nm的切片。将单个部分从部分带中分离出来，并将单个部分漂浮在水浴中。</li>
	<li>将一滴ddH2O滴到金纳米颗粒涂层盖玻片的中心。使用样品环拾取一个截面，并将样品环倒置到水滴表面。取下样品环，将部分留在水滴表面。</li>
	<li>将盖玻片风干几分钟。用记号笔在盖板另一侧的部分周围画一个环。</li>
</ol>5.光学显微成像
<ol><li>荧光回收 注意：工作流程如 图 3A 所示。<ol><li>将 10 μL 封固缓冲液（参见 材料表）添加到盖玻片上的超薄部分上。将一个新的干净盖玻片放在安装缓冲器上。 注意：封装缓冲液的关键成分是DABCO，用于回收荧光蛋白的荧光。这对于荧光恢复非常重要。</li>
		<li>将这两个盖玻片与超薄切片一起放入成像室中（参见 材料表）。确保带有拉出环的盖玻片位于顶部。</li>
	</ol></li>
	<li>收集导航地图。<ol><li>在倒置荧光显微镜 的明场成像模式 下，使用绘制的环形作为标记物找到切片（参见 材料表）。移动到该部分的一角。打开白光并拍摄 明场图像。</li>
		<li>关闭白灯。打开激光 （561 nm） 并拍摄相同视场 （FOV） 的荧光图像。</li>
		<li>关闭激光。移动到相邻的 FOV，确保这两个 FOV 有 10% 的重叠。获取第二个 FOV 的明场图像和荧光图像。</li>
		<li>重复此操作，直到覆盖整个部分。记录可用于拼接 FOV 的成像路径。 注意：为了获得更高质量的图像，我们建议使用共聚焦显微镜。</li>
	</ol></li>
	<li>对感兴趣的单元格进行成像。<ol><li>使用导航地图轻松定位感兴趣的单元格。打开激光（561nm）并拍摄300帧感兴趣细胞的荧光图像。关闭激光。</li>
		<li>打开白光并拍摄连续的明场图像（~100 帧）。</li>
		<li>移动到另一个感兴趣的单元格。</li>
	</ol></li>
</ol>6. 电镜成像的准备
注意：工作流程如 图 3B 所示。
<ol><li>将超薄部分转移到槽栅。<ol><li>用ddH2O填充玻璃罐。</li>
		<li>从腔室中取出盖玻片，并用镊子将两个盖玻片分开。夹紧截面所在的盖玻片，洗掉安装缓冲液，然后风干。</li>
		<li>使用单面刀片在超薄截面周围划一个#，并在#的每个角上滴入10μL 12%氢氟酸（参见材料表）。将盖玻片慢慢放入水中。 注意：氢氟酸与玻璃反应，更容易从盖玻片上去除毛状膜。氢氟酸有毒。在戴手套的化学罩中使用。与皮肤接触需要立即就医。</li>
		<li>将毛状薄膜和超薄切片一起漂浮在水面上。在截面上放置一个未涂层的槽网格，以捕获网格中心的截面。</li>
		<li>用封口膜覆盖载玻片（参见 材料表），然后用 pioloform 薄膜拾取网格。在室温下风干网格。</li>
	</ol></li>
	<li>切片染色<ol><li>用 2% UA 和 Sato 的三重领先优势染色该部分。有关详细信息，请参阅之前发布的协议2。</li>
	</ol></li>
</ol>7. 图像分析
<ol><li>拼接导航地图。<ol><li>查找原始数据的位置。打开软件ImageJ（见 材料表）拼接导航图。</li>
		<li>点击 插件 |缝合 |网格/系列拼接 |（类型）网格：按列排列的蛇 |（订单）向上和向左 （根据拍摄顺序） |设置切片大小 |选择数据路径 |设置文件名 |选中显示融合。观察结果，这是一个由不同 FOV 的几个明场图像组成的导航图。</li>
		<li>使用相同的方法拼接荧光导航图。</li>
	</ol></li>
	<li>将明场图像与荧光图像合并。<ol><li>在软件 ImageJ 中，单击 “文件”|”导入 |图像序列 ，用于导入感兴趣单元的连续明场图像。</li>
		<li>点击 图片 |堆栈 |Z投影 |求和切片 ，用于获取序列明场图像的总和强度投影 （SIP） 图像。</li>
		<li>点击 编辑 |反转 将明场的SIP图像反转，使金纳米粒子的信号变成明亮的白点。</li>
		<li>在 ImageJ 中，单击“ 文件”|”导入 |Image Sequence 导入连续的 FM 图像。</li>
		<li>点击 图片 |堆栈 |Z投影 |求和切片 以获取顺序 FM 映像的 SIP 映像。</li>
		<li>点击 图片 |颜色 |合并通道 以将 SIP 明场图像与 SIP FM 图像合并为复合图像。</li>
		<li>金纳米颗粒是绿色的，荧光蛋白是红色的。点击 图片 |类型 |RGB 颜色 将合成图像的格式转换为 RGB。</li>
	</ol></li>
</ol>8. 电磁成像
<ol><li>将网格放在样品支架上，保持网格的截面朝上。</li>
	<li>使用导航图，通过到超薄切片右梯形的四个不同角的四个相对距离，在电子显微镜下大致识别相应细胞的位置。</li>
	<li>使用金纳米颗粒确认细胞。</li>
	<li>使用透射电子显微镜 （TEM） 以不同的放大倍率（4,200 倍、6,000 倍或 8,200 倍）拍摄 EM 图像（参见 材料表）。</li>
</ol>9. FM图像与EM图像的配准
<ol><li>打开注册软件（参见 材料表）。在搜索窗口中键入 easyCLEMv014。单击 easyCLEMv0 打开 easyCLEMv0。点击 图像/序列 |打开 以将 EM 图像和合成图像导入软件面板。</li>
	<li>在 EasyCLEMv0 窗口中，单击 2D （x，y，[T]） 以选择 非刚性（2D 或 3D） 作为 对齐模式。</li>
	<li>在 EasyCLEMv0 窗口中，单击 “选择将要转换和调整大小的图像”（可能是 FM） 右侧的下拉框以选择 “复合 （RGB）color.tif。然后，单击右侧的下拉框 选择不会被修改的图像（可能是 EM） 以选择 EMimage. tif。</li>
	<li>单击 “开始 ”开始注册。</li>
	<li>在名为 EMimage. tif 的 EM 图像窗口中，单击一个金纳米颗粒，将 点 1 放在金纳米颗粒上。在名为 “复合（RGB）颜色.tif的FM图像窗口中，单击相应的金纳米颗粒，将 点1 放在金纳米颗粒上。</li>
	<li>单击 “更新变换 ”以确认LM信号与金纳米颗粒的EM信号。</li>
	<li>在名为EMimage. tif的EM图像窗口中重复上述操作 ， 将LM信号与同一金纳米颗粒的EM信号逐一匹配。</li>
	<li>单击 “停止 ”以完成注册。</li>
	<li>图像对齐后，单击 “叠加 图像”，然后单击 “图像/序列”|”另存为 以导出包含四个通道图像（红色、绿色、蓝色、灰色）的图像堆栈。</li>
	<li>打开 ImageJ 并单击 “文件”|”打开 以导入 Overlayed 图像。点击 图片 |堆栈 |Stack to Images 将 Overlayed 图像分成四个通道图像。</li>
	<li>点击 图片 |颜色 |合并通道 将来自三个通道（红色、绿色和灰色）的图像合并到一个合成图像中。</li>
	<li>点击 图片 |类型 |RGB 颜色 将合成图像转换为 CLEM 图像。</li>
</ol>

同时保存荧光和超微结构的新方案

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E., 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=Comprehensive+dual-+and+triple-feature+intersectional+single-vector+delivery+of+diverse+functional+payloads+to+cells+of+behaving+mammals.">Comprehensive dual- and triple-feature intersectional single-vector delivery of diverse functional payloads to cells of behaving mammals.</a> Neuron. 107 (5), 836-853 (2020).</li><li>Shcherbo, D., 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=Far-red+fluorescent+tags+for+protein+imaging+in+living+tissues.">Far-red fluorescent tags for protein imaging in living tissues.</a> Biochem J. 418 (3), 567-574 (2009).</li><li>Shen, Y., Chen, Y., Wu, J., Shaner, N. C., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+mCherry+variants+with+long+Stokes+shift,+red-shifted+fluorescence,+and+low+cytotoxicity.">Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity.</a> PLoS One. 12 (2), e0171257 (2017).</li><li>Ai, H. W., Olenych, S. G., Wong, P., Davidson, M. W., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hue-shifted+monomeric+variants+of+Clavularia+cyan+fluorescent+protein:+identification+of+the+molecular+determinants+of+color+and+applications+in+fluorescence+imaging.">Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging.</a> BMC Biol. 6 (13), 1741 (2008).</li><li>Ogoh, K., 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=Dual-color-emitting+green+fluorescent+protein+from+the+sea+cactus+Cavernularia+obesa+and+its+use+as+a+pH+indicator+for+fluorescence+microscopy.">Dual-color-emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy.</a> Luminescence. 28 (4), 582-591 (2013).</li><li>Bindels, D. S., 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=mScarlet:+a+bright+monomeric+red+fluorescent+protein+for+cellular+imaging.">mScarlet: a bright monomeric red fluorescent protein for cellular imaging.</a> Nat Methods. 14 (1), 53-56 (2017).</li><li>Xu, P., Xu, T., Zhang, M., Fu, Z., He, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+Epon-embedding-based+correlative+super-resolution+light+and+electron+microscopy.">A protocol for Epon-embedding-based correlative super-resolution light and electron microscopy.</a> Research Square. , (2019).</li><li>Johnson, E., Kaufmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+photoswitching+ability+of+standard+fluorescent+proteins+for+correlative+in-resin+super-resolution+and+electron+microscopy.">Preserving the photoswitching ability of standard fluorescent proteins for correlative in-resin super-resolution and electron microscopy.</a> Methods Cell Biol. 140, 49-67 (2017).</li><li>Zhou, S., 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=Liquid-liquid+phase+separation+mediates+the+formation+of+herpesvirus+assembly+compartments.">Liquid-liquid phase separation mediates the formation of herpesvirus assembly compartments.</a> J Cell Biol. 222 (1), 17 (2023).</li><li>Tao, C. L., 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=Differentiation+and+characterization+of+excitatory+and+inhibitory+synapses+by+cryo-electron+tomography+and+correlative+microscopy.">Differentiation and characterization of excitatory and inhibitory synapses by cryo-electron tomography and correlative microscopy.</a> J Neurosci. 38 (6), 1493-1510 (2018).</li></ol>

作者没有利益冲突需要声明。

这里介绍的方案是一种通用成像方法，它可以结合光学显微镜 （LM） 中靶蛋白的定位信息和电子显微镜 （EM） 中靶蛋白周围的背景6。由于目前荧光蛋白的局限性，广泛使用的方法是预嵌入相关光和电子显微镜 （CLEM），这意味着 LM 成像是在 EM 样品制备之前完成的。几乎所有现有的荧光蛋白都可以在预包埋CLEM中进行检查。然而，由于不可避免的失真和收缩，最终图像的精确对?...

荧光显微镜（FM）可用于获得靶蛋白的定位和分布。然而，靶蛋白周围的背景丢失了，这对于彻底研究靶蛋白至关重要。电子显微镜 （EM） 具有最高的成像分辨率，可提供多个亚细胞细节。然而，EM缺乏靶标标记。通过将FM采集的荧光图像与EM采集的灰度图像精确合并，相关光电子显微镜（CLEM）可以将这两种成像模式1,2,3,4获得的信息结合起来。
CLEM 是荧光和超微结构之间的权衡 1.由于目前荧光蛋白和传统电镜样品制备程序的局限性，特别是使用锇酸（OsO4）和疏水性树脂（如Epon），超微结构总是影响荧光5。OsO4 是 EM 样品制备中不可或缺的试剂，用于提高 EM 图像的对比度。与其他包埋树脂相比，Epon 在超微结构保存和切片性能方面更胜一筹5。然而，在OsO4 和Epon embedding6处理后，没有荧光蛋白可以保留荧光信号。为了克服荧光蛋白的局限性，开发了预包埋 CLEM，其中 FM 成像在 EM 样品制备之前完成 6。然而，预嵌入 CLEM 的缺点是 FM 和 EM 图像之间的配准不精确5.
相反，嵌入后CLEM方法在EM样品制备后进行FM成像，其配准精度可达6-7nm 5,6。为了保留荧光蛋白的荧光，使用极低浓度的OsO4（0.001%）3或高压冷冻（HPF）和冷冻取代（FS）EM制备方法4,7，以牺牲超微结构受损或EM图像的对比度为代价。尽管使用甲基丙烯酸缩水甘油酯作为包埋树脂5，但mEos4b的发展极大地促进了包埋后CLEM的进展。随着 mEosEM 的发展，该 mEosEM 可以在 OsO4 染色和 Epon 包埋中存活下来，首次实现了 Epon 后包埋超分辨率 CLEM，同时保持了荧光和超微结构6.在 mEosEM 之后，开发了几种可以在 OsO4 染色和 Epon 包埋中存活的荧光蛋白 8,9,10,11。这极大地促进了CLEM的发展。
Epon 嵌入后 CLEM 有三个关键方面。首先是荧光蛋白，在EM样品制备后应保持荧光信号。根据我们的经验，mScarlet优于其他报道的荧光蛋白。第二个是如何在EM成像中找到通过FM成像成像的相同细胞。为了解决这个问题，我们改进了这一步的程序，以便人们可以很容易地找到目标细胞。最后一种是将 FM 图像与 EM 图像对齐的方法。在这里，我们详细介绍了 FM 和 EM 图像之间的配准。在该协议中，我们在 VGLUT2 神经元中表达 mScarlet，并证明 mScarlet 可以使用 Epon 嵌入后 CLEM 靶向二级溶酶体。我们提供了 Epon 嵌入后 CLEM 的分步细节，而不会影响荧光和超微结构。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 M Phosphate Buffer (PB)</td>
			<td></td>
			<td></td>
			<td>NaH2PO4 &#183; 2H2O+Na2HPO4 &#183; 12H2O</td>
		</tr>
		<tr>
			<td>0.2 M Tris-Cl (pH 8.5)</td>
			<td>Shanghai yuanye Bio-Technology</td>
			<td>R26284</td>
			<td></td>
		</tr>
		<tr>
			<td>25% Glutaraldehyde (GA)</td>
			<td>Alfa Aesar</td>
			<td>A17876</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr>
			<td>Abbelight 3D</td>
			<td>Nanolnsights</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>SCR</td>
			<td>10000418</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>J&#38;K Scientific</td>
			<td>335213</td>
			<td></td>
		</tr>
		<tr>
			<td>BioPhotometer D30</td>
			<td>eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaning buffer of cover glasses</td>
			<td></td>
			<td></td>
			<td>50 mL Ammonium hydroxide, 50 mL Hydrogen peroxide, 250 mL H2O</td>
		</tr>
		<tr>
			<td>Coverglass</td>
			<td>Warner</td>
			<td>64-0715</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO&#160;</td>
			<td>Sigma</td>
			<td>290734</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>SPI company</td>
			<td>GS02827</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr>
			<td>Desktop centrifuge</td>
			<td>WIGGENS</td>
			<td>MINICEN 10E</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond knife</td>
			<td>DiATOME</td>
			<td>MX6353</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>SPI company</td>
			<td>GS02823</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr>
			<td>DNA transfection reagent</td>
			<td>Thermo Fisher&#160;</td>
			<td>2696953</td>
			<td>Lipofectamine 3000 Transfection Kit</td>
		</tr>
		<tr>
			<td>Epon 812&#160;</td>
			<td>SPI company</td>
			<td>GS02659</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>SCR</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji image J</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixative solution&#160;</td>
			<td></td>
			<td></td>
			<td>4% PFA+0.25% GA+0.02 M PB</td>
		</tr>
		<tr>
			<td>Formvar</td>
			<td>Sigma</td>
			<td>9823</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>SCR</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold nanoparticles</td>
			<td>Corpuscular</td>
			<td>790120-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient resin</td>
			<td></td>
			<td></td>
			<td>Acetone to resin 3:1, 1:1, 1:3</td>
		</tr>
		<tr>
			<td>Hydrofluoric acid</td>
			<td>SCR</td>
			<td>10011118</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>SCR</td>
			<td>10011218</td>
			<td></td>
		</tr>
		<tr>
			<td>ICY (https://icy.bioimageanalysis.org/about/)</td>
			<td></td>
			<td></td>
			<td>Easy CLEMv0 Plugin</td>
		</tr>
		<tr>
			<td>Imaging chamber</td>
			<td>Thermo Fisher&#160;</td>
			<td>A7816</td>
			<td></td>
		</tr>
		<tr>
			<td>Large gelatin capsules</td>
			<td>Electron Microscopy Sciences</td>
			<td>70117</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting buffer</td>
			<td></td>
			<td></td>
			<td>Mowiol 4-88, Glycerol, 0.2 M Tris-Cl (pH 8.5), DABCO</td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma</td>
			<td>9002-89-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#160;&#382;12H2O</td>
			<td>SCR</td>
			<td>10020318</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4&#160;&#382;2H2O</td>
			<td>SCR</td>
			<td>20040718</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>SPI company</td>
			<td>GS02828</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr>
			<td>Oligonucleotide primers</td>
			<td>Takara Biomedical Technology (Beijing)</td>
			<td></td>
			<td>Three oligonucleotides primers were used to detect Vglut2-ires-Cre and wild-type simultaneously. The primers 5,-ATCGACCGGTAATGCAGGCAA-3, and 5,-CGGTACCACCAAATCTTACGG-3, aimed to detect Vglut2-ires-Cre. The primers&#160; 5,-CGGTACCACCAAATCTTACGG-3, and 5,-CATGGTCTGTTTTGAATTCAG-3, aimed to detect wild-type.</td>
		</tr>
		<tr>
			<td>Oscillating microtome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>SCR</td>
			<td>L01210302</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr>
			<td>OsO4 solution</td>
			<td></td>
			<td></td>
			<td>1% Osmium tetroxide+1.5% K4Fe (CN)6&#183;3H2O</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Amcor</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>SCR</td>
			<td>80096618</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr>
			<td>Perfusion buffer</td>
			<td></td>
			<td></td>
			<td>4% PFA+0.1 M PB</td>
		</tr>
		<tr>
			<td>Pioloform</td>
			<td>Sigma</td>
			<td>63148-65-2</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr>
			<td>Poly-L-lysine&#160;</td>
			<td>Sigma</td>
			<td>25986-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanide (K4Fe (CN)6&#183;3H2O)&#160;</td>
			<td>SCR</td>
			<td>10016818</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Merck</td>
			<td>S2771</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handles</td>
			<td>Merck</td>
			<td>S2896-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>OLYMPUS</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Transgenic mice</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td>Vglut2-ires-Cre mice (strain: 129S6/SvEvTac) were housed in standard conditions (25 &#176;C, a 12 h light/dark cycle, with water and food given ad libitum. Male and Female mice were used at 2&#8211;3 months old, weight range 20-30 g.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Transmission electron microscope (TEM)</td>
			<td>FEI</td>
			<td></td>
			<td>TECNAL G2</td>
		</tr>
		<tr>
			<td>UA solution (2% UA)</td>
			<td></td>
			<td></td>
			<td>Aqueous solution</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>LEICA EM UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate (UA)</td>
			<td>TED PELLA</td>
			<td>19481</td>
			<td>Hazardous chemical</td>
		</tr>
	</tbody>
</table>

epon post embedding correlative light and electron microscopy

相关光电子显微镜（CLEM）是一种综合显微镜，它结合了荧光显微镜（FM）提供的定位信息和电子显微镜（EM）获取的细胞超微结构的背景。CLEM是荧光和超微结构之间的权衡，通常，超微结构会影响荧光。与其他亲水性包埋树脂（如甲基丙烯酸缩水甘油酯、HM20或K4M）相比，Epon在超微结构保存和切片性能方面更胜一筹。此前，我们已经证明mEosEM可以在四氧化锇固定和Epon包埋中存活。使用mEosEM，我们首次实现了EPON后嵌入CLEM，它同时保持荧光和超微结构。在这里，我们提供了有关 EM 样品制备、FM 成像、EM 成像和图像对齐的分步详细信息。我们还改进了在 EM 成像期间识别 FM 成像的相同细胞的程序，并详细说明了 FM 和 EM 图像之间的配准。我们相信，在传统的电磁设施中，按照这种新协议，可以很容易地实现嵌入相关光和电子显微镜的Epon后。

Fluorescence microscopy (FM) can be used to obtain the localization and distribution of the target protein. However, the context that surrounds the target protein is lost, which is crucial for investigating the target protein thoroughly. Electron microscopy (EM) has the highest imaging resolution, providing several subcellular details. Nevertheless, EM lacks target labeling. By accurately merging the fluorescence image taken by FM with the gray image acquired by EM, correlative light and electron microscopy (CLEM) can combine the information obtained by these two imaging modes1,2,3,4.
CLEM is a trade-off between fluorescence and the ultrastructure1. Because of the limitations of current fluorescent proteins and the traditional EM sample preparation procedures, especially the use of osmic acid (OsO4) and hydrophobic resins such as Epon, the ultrastructure always compromises fluorescence5. OsO4 is an indispensable reagent in EM sample preparation, which is used to improve the contrast of EM images. Compared with other embedding resins, Epon is superior in ultrastructure preservation and sectioning properties5. However, no fluorescent proteins can retain the fluorescence signal after the treatment of OsO4 and Epon embedding6. To overcome the limitations of fluorescent proteins, pre embedding CLEM was developed, in which FM imaging is done before EM sample preparation6. However, the drawback of pre embedding CLEM is the imprecise registration between the FM and the EM images5.
On the contrary, post embedding CLEM method performs the FM imaging after the EM sample preparation, the registration accuracy of which can reach 6-7 nm5,6. To retain the fluorescence of fluorescent proteins, very low concentrations of OsO4 (0.001%)3 or the high-pressure frozen (HPF) and freeze substitution (FS) EM preparation methods4,7 have been used at the expense of compromised ultrastructure or the contrast of the EM image. The development of mEos4b greatly promotes the progress of post embedding CLEM, although glycidyl methacrylate is used as the embedding resin5. With the development of mEosEM, which can survive the OsO4 staining and Epon embedding, Epon post embedding super-resolution CLEM was achieved for the first time, maintaining the fluorescence and ultrastructure simultaneously6. After mEosEM, several fluorescent proteins that can survive the OsO4 staining and the Epon embedding were developed8,9,10,11. This greatly promotes the development of CLEM.
There are three key aspects to Epon post-embedding CLEM. The first is the fluorescent protein, which should maintain the fluorescent signal after EM sample preparation. According to our experience, mScarlet is superior to other reported fluorescent proteins. The second is how to find the same cell imaged by FM imaging in EM imaging. To solve this problem, we improve the procedure for this step so that one can readily find the targeted cell. The last is the method to align the FM image with the EM image. Here, we detail the registration between the FM and the EM images. In this protocol, we express mScarlet in VGLUT2 neurons and demonstrate that mScarlet can target secondary lysosomes using Epon post-embedding CLEM. We provide step-by-step details for Epon post-embedding CLEM, without compromising the fluorescence and the ultrastructure.

作者聚焦：相关光学和电子显微镜与荧光蛋白保存的进步

Epon Post 嵌入相关光和电子显微镜

Correlative light and electron microscopy is a completely stable microscopy technique, which can combine the localization information provided by fluorescent microscopy and is the ultra structure by electron microscopy. However, the mentality of this technique is to preserve the fluorescence of after you have a simple preparation. Using this fluorescent protein, we can achieve correlative light and the electron microscope which can maintain fluorescence and ultra structure simultaneously.

先前的报道表明，mScarlet可以靶向溶酶体15。在该方案中，使用立体定位仪器将表达 mScarlet 的 AAV （rAAV-hSyn-DIO-mScarlet-WPRE-pA） 注射到 Vglut2-ires-cre 小鼠大脑的 M1 （ML： ±1.2 AP： +1.3 DV： -1.5） 中。按照上述协议，最终的相关图像如图4A所示。使用金纳米颗粒（绿点）作为基准标记，FM图像可以与EM图像精确对齐。如EM图像（图4C，F</s...

Watch this Scientific Journal Video about Epon Post Embedding Correlative Light and Electron Microscopy at JoVE.com

我们提出了使用称为 mScarlet 的荧光蛋白进行 Epon 嵌入后相关光学和电子显微镜的详细方案。这种方法可以同时保持荧光和超微结构。该技术适用于各种生物应用。

Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence ...

相关光电显微镜是一种完全稳定的显微镜技术，它既能结合荧光显微镜提供的定位信息，又能结合电子显微镜的超结构。但是，这种技术的心态是在您进行简单准备后保留荧光。使用这种荧光蛋白，我们可以实现相关的光和电子显微镜，可以同时保持荧光和超结构。

epon-post-embedding-correlative-light-and-electron-microscopy

Research

JoVE Journal

Biology

Epon Post Embedding Correlative Light and Electron Microscopy

841 次观看.  Fujian Medical University. 相关光电显微镜是一种完全稳定的显微镜技术，它既能结合荧光显微镜提供的定位信息，又能结合电子显微镜的超结构。但是，这种技术的心态是在您进行简单准备后保留荧光。使用这种荧光蛋白，我们可以实现相关的光和电子显微镜，可以同时保持荧光和超结构。

视频: 作者聚焦：相关光学和电子显微镜与荧光蛋白保存的进步

Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence microscopy (FM) and the context of cellular ultrastructure acquired by electron microscopy (EM). CLEM is a trade-off between fluorescence and ultrastructure, and usually, ultrastructure compromises fluorescence. Compared with other hydrophilic embedding resins, such as glycidyl methacrylate, HM20, or K4M, Epon is superior in ultrastructure preservation and sectioning properties. Previously, we had demonstrated that mEosEM can survive osmium tetroxide fixation and Epon embedding. Using mEosEM, we achieved, for the first time, Epon post embedding CLEM, which maintains the fluorescence and the ultrastructure simultaneously. Here, we provide step-by-step details about the EM sample preparation, the FM imaging, the EM imaging, and the image alignment. We also improve the procedures for identifying the same cell imaged by FM imaging during the EM imaging and detail the registration between the FM and EM images. We believe one can easily achieve Epon post embedding correlative light and electron microscopy following this new protocol in traditional EM facilities.

We present a detailed protocol for Epon post-embedding correlative light and electron microscopy using a fluorescent protein called mScarlet. This method can maintain the fluorescence and the ultrastructure simultaneously. This technique is amenable to a wide variety of biological applications.