这项工作得到了国家卫生研究院的资助, K08 NS061039-01A2, 神经病学研究和 #38; 发现, 和密歇根大学 Taubman 医学研究所的 a。这项工作使用的形态学和图像分析核心的密歇根糖尿病研究中心, 由国家卫生研究院资助 5P90 DK-20572 来自国家糖尿病和消化系统和肾脏疾病研究所。作者要感谢 j. 鲁滨逊单身人士和 a. 戈登？史密斯 (犹他大学) 慷慨捐献人体皮肤样本。

在犹他州糖尿病中心 (盐湖城), 从一个大型社区初级保健网络招募的受试者中获得了皮肤穿孔活检。这项研究得到了密歇根大学机构审查委员会的批准, 并遵守了《赫尔辛基宣言》的宗旨。在测试之前, 从每个主题获得书面知情同意. 
 1. 荧光免疫组化 <ol> <li> 为 intraepidermal 神经纤维免疫组织化学制备穿孔活检: <ol> <li> 由医务人员进行3毫米皮肤活检, 并将整个活检放在1。5磨和 #39 s 固定液 (2% 甲醛, 0.3% 饱和苦味酸酸在磷酸盐缓冲盐水 (PBS), pH 7.4) 在4和 #176; C 过夜. </li>
		<li> 在 PBS 中用30% 蔗糖的溶液冲洗样品, 4 和 #176; C 为16-24 小时或直到样品下沉. </li>
		<li> 使用 cryomold 在最佳切削温度复合 (OCT) 中嵌入样本。放置整个 3 mm 活检与表皮朝下在模具和填充模具约2毫升的 OCT. 在粉碎的干冰上冻结模具。存储在-80 和 #176; C 直到准备使用. </li>
		<li> 切割50和 #181; m 厚剖面使用低温和存储在单个井的一个 96-井板使用180和 #181; L 防冻液每井 (30% 乙二醇, 30% 甘油在 PBS)。以下方向为96井板的8口井。从每一个活检网站 200-300 和 #181 的污点切片; 我远离彼此. </li>
	</ol> </li> </ol> 日 1: <ol start="2"> <li> 对地层角质的非特异标记: <ol> <li> 标签 96-好板如 图 1 中所示. </li>
		<li> 吸管150和 #181; L 股票信号增强剂解决方案, 以减少次级抗体的非特异性结合 34 , 35 到每个井。使用接种回路将部分转移到信号增强器解决方案中. 
		注意: 在使用组织时要小心, 避免损坏或撕裂组织切片。保持在信号增强解决方案在室温下的一个平面摇杆30分钟. </li>
		<li> 通过添加150和 #181, 在96孔板的2和3行中准备冲洗井, 将1x 磷酸酯缓冲盐水 (PBS) 放入每个井中. </li>
		<li> 小心地将部分转移到2行, 并在室温下用 1x PBS 冲洗10分钟. </li>
		<li> 在室温下第二次在 1x PBS 中冲洗10分钟 (3 行). </li>
	</ol> </li> <li> 准备5% 牛血清白蛋白 (bsa) 阻止解决方案: <ol> <li> 在 100 M PBS 中准备5% 个包含 5% BSA 和0.3% 卫一 X 0.1 (TX-100) 的拦截解决方案 (参见 表 1 ), 而部分则在孵化信号增强解决方案。波塞军不容易进入解决方案。涡旋溶液直到 BSA 完全溶解. </li>
		<li> 在96井板的4排中, 通过添加150和 #181 来准备堵塞井; 5% BSA 将溶液插入每个井中. </li>
		<li> 将剖面转换为 5% bsa 阻断溶液的单个井, 并将 5% bsa 的 1-2 h 在室温下的平板摇杆中孵育剖面. </li>
	</ol> </li> <li> 准备 1% bsa 冲洗溶液和主抗体稀释液: <ol> <li> 准备1% 冲洗解决方案, 其中包含 1% bsa 和 0.3% TX-100 0.1 M PBS (见 表 2 ), 而节是在 5% BSA 阻断溶液中孵化。波塞军不容易进入解决方案。涡旋溶液直到 BSA 完全溶解. </li>
		<li> 在 1% bsa 冲洗液中稀释主抗体, 而切片则在 5% bsa 阻断溶液中孵化。
		<ol> <li> 使1500和 #181; 主抗体溶液 L, 并在5行中的每个井中添加. </li>
			<li> 稀释主要抗体: 使用神经特异的标签, 兔多克隆 anti-protein 基因产品 9.5 (PGP9.5) 在 1:1, 000。使用线粒体特异的标签, 小鼠单克隆 anti-pyruvate 脱氢酶 E2/E3bp 抗体 (人) 在1:100 在 1% BSA 漂洗溶液. </li>
		</ol> </li> </ol> </li> <li> 准备主抗体: <ol> <li> 吸管150和 #181; 在96个井板的5排中每一个井的主抗体 L. </li>
		<li> 使用循环工具, 将阻塞解决方案 (行 4) 中的部分传输到包含主抗体的行5中. </li>
		<li> 用膜把盘子紧紧地裹起来以防止它干燥. </li>
		<li> 将样品放在室温为1小时的平摇杆上, 然后在4和 #186 上孵育样品; C 在一夜之间的平摇杆上. </li>
	</ol> </li> </ol> 日 2: <ol start="6"> <li> 冲洗样品: <ol> <li> 在 96-井板的6、7和8行中准备冲洗井, 并加入150和 #181; 1% BSA 的 L, 每井冲洗溶液. </li>
		<li> 将剖面转换为前 1% BSA 冲洗液 (6 排) 并在室温下孵育1小时。重复漂洗两次以上的 1% BSA 冲洗解决方案在7和8行每在室温下1小时. </li>
	</ol> </li> <li> 在 1% bsa 漂洗解决方案中稀释二级抗体: <ol> <li> 使1500和 #181; 二级抗体溶液 L 在最后漂洗的 1% bsa 漂洗溶液 (列 8) 中孵化。
		<li> 稀释二级抗体: 用于 PGP9.5 (绿色荧光共轭山羊兔抗体, 1:1000), 用于 (红色荧光共轭山羊 anti-mouse, 1:1, 000) 在 1% BSA 漂洗溶液中. </li>
	</ol> </li> <li> 准备二级抗体: <ol> <li> 吸管150和 #181; 二级抗体升至96井板的9排. </li>
		<li> 将 1% BSA 冲洗液 (8 排) 的部分轻轻地转到二级抗体井 (9 排). </li>
		<li> 使用膜, 将钢板紧紧地包裹起来, 防止其干燥。用铝箔盖。将样品放在室温为1小时的平摇杆上, 然后在4和 #730 上孵育样品; C 在一夜间平的摇椅上. </li>
	</ol> </li> </ol> 天 3: <ol start="9"> <li> 通过0.22 和 #181 筛选来准备干净的 1x pbs; m 过滤器: <ol> 吸管150和 #181; 过滤后的 1x pbs 为10行、11和 12. <li>
		<li> 将样品转移到10行, 并在室温下在 1x PBS 中冲洗1小时。用铝箔覆盖96井板, 在冲洗时将其放在平摇杆上。重复过滤的 1x PBS 冲洗两次1小时, 每在室温在11和12行. </li>
	</ol> </li> <li> 准备用于安装组织切片的显微镜幻灯片: <ol> <li> 放置50和 #181; 在幻灯片上筛选 1x PBS 的 L. </li>
		<li> 将 1x PBS (12 行) 中的部分传输到50和 #181; 我掉了小心地放置在 PBS 下降的部分, 展开组织, 并轻轻地铺平它到玻璃幻灯片。用玻璃吸管除去多余的 PBS, 避免稀释安装试剂。不要触摸玻璃灯泡的部分. </li>
		<li> 吸管 1-2 滴安装试剂, 其中含有 DAPI 直接在显微镜幻灯片上的部分的顶部, 使用注意不要扰乱该节的方向。轻轻地放置一个50毫米 x 24 毫米 #1. 5 显微镜玻璃片在该部分. </li>
		<li> 清除在放置片时形成的气泡, 并将多余的液体从片的边缘擦除。准备一个新的幻灯片, 并重复安装公关每节 ocess. </li>
		<li> 通过在室温下一夜之间将幻灯片放置在黑暗中来固化/干燥安装介质。将幻灯片传输到4和 #176; c 用于短期 (1-2 周) 或-20 和 #176; c 用于长期存储 (大于2周). 
		注意: 阴性对照在阶梯1.4.2.2 中省略了 PGP9.5 的主要抗体, 并且在表皮中没有显示出可区分的神经或线粒体标记。对该抗体进行了阳性对照, 以证明它标记了所有的线粒体 (未显示数据)。对培养的原代小鼠背根神经节神经元进行了阳性对照, 用杆状病毒转荧光蛋白 (GFP) 信号对线粒体进行标记, 然后用抗体和红色荧光固定和染色二级抗体。所有表达 GFP 的线粒体都被 co-labeled 为免疫组织化学的红色标签 (未显示数据). </li>
	</ol> </li> </ol> 2。共焦成像 <ol> <li> 执行共聚焦成像: <ol> <li> 使用激光扫描共聚焦显微镜收集图像, 40X 油浸 (1.25 数字孔径 (非)) 目标倒置显微镜。
		<ol> <li> 在每个焦平面上依次获取荧光信号: 原子核: 激发和 #955; = 405 nm, 光谱发射过滤器和 #955; = 420-480 nm 神经纤维: 激发和 #955; = 488 nm, 光谱发射过滤器和 #955; = 505-560 nm Mitochondria: 激发和 #955; = 543 nm, 光谱发射过滤器和 #955; = 606-670 nm </li> </ol> </li> <li> 在显微镜软件中输入以下扫描参数: 600 Hz 的扫描速率, 2 帧平均和缩放 2.2; 12 位强度分辨率 (4096 灰度级别)。
		<ol> <li> 设置用于优化横向分辨率的显微镜软件 (扫描分辨率 = 1024 x 1024) 和轴向分辨率/光学切片 (共焦孔径 = 1 通风单元 (AU), z 步尺寸为 210 nm). 
			注: 产生的 XYZ 分辨率为 172.2 nm x 172.2 nm x 210 nm, 图像大小为176.1 和 #181; m x 176.1 和 #181; m x 30-50 和 #181; m. </li>
		</ol> </li> <li> 激活对神经信号的实时扫描 (绿色荧光), 并调整 z 焦点控制, 以在显微镜软件中查找和设置包含该组织部分内的神经信号的上、下焦平面。总 z 范围通常是30-50 和 #181; m 为50和 #181; m 组织部分. </li>
		<li> 在实时扫描过程中, 使用显微镜软件旋转扫描域, 使表皮在图像中处于水平或垂直位置. </li>
		<li> 分别扫描每个信号, 并调整探测器 (光电倍增管、PMT) 的电压和偏移量以最小化/删除任何超过或低于饱和像素. 
		注: 扫描时间与上述参数约 20-40 分钟, 取决于 z 切片的数量. </li>
	</ol> </li> </ol> 3. 3D 人 Intraepidermal 神经纤维内线粒体的可视化和分析 <ol> <li> 隔离3D 表皮: <ol> <li> 复制原始图像并使用最大强度图像的投影 (扩展焦点视图) 来识别和隔离表皮. </li>
		<li> 使用感兴趣的区域工具沿表皮的上下边缘进行追踪, 以去除不需要的区域, 如 intraepidermal 神经纤维缺乏的角质层和真皮。裁剪到此选定内容. </li>
	</ol> </li> <li> 使用神经和线粒体荧光信号的反褶积: 注意: 反褶积有助于恢复荧光信号的完整性。此协议中使用的恢复称为盲反褶积, 因为它使用图像中的荧光信号来确定信号从其原始源 (点扩展函数) 传播了多少。该过程通过将信号重新分配回原位置来提高信号分辨率。
	<ol> <li> 计算用于绿色荧光信号 (绿色荧光) 的点扩展函数 (聚砜), 其参数如下: <ol> <li> 设置计算的聚砜为共聚焦。将介质折射率设置为 1.515, 数值孔径为1.25。将探测器针孔设置为1通风装置 (a.u)。将激光激励波长设置为 488 nm, 发射波长为 515 nm. </li>
		</ol> </li> <li> 使用以下参数计算红色荧光线粒体信号 (红色荧光) 的聚砜: <ol> <li> 将计算的聚砜设置为共聚焦。将介质折射率设置为 1.515, 数值孔径为1.25。将探测器针孔设置为 1 AU。将激光激励波长设置为 543 nm, 发射波长为 617 nm. </li>
		</ol> </li> <li> 通过使用上面列出的相应 PSFs 和迭代恢复功能设置为100% 的置信度和10周期的迭代限制, 对神经和线粒体荧光信号进行反褶积优化. </li>
	</ol> </li> <li> 创建特定于神经的曲面: <ol> <li> 使用和 #34; 创建曲面和 #34; 工具, 使一个固体表面的神经从积绿色荧光二次标记的 PGP9.5 识别神经. </li>
		<li> 取消选中和 #34; 平滑和 #34; 特征并使用绝对强度特性来设置神经信号的阈值, 因为它比背景荧光明显亮. </li>
		<li> 使用绝对强度特征来设置神经信号的阈值, 因为它比背景荧光明显亮。设置阈值足够低, 以准确识别神经. </li>
		<li> 根据大小筛选出小的、non-nerve 的曲面. 
		注: 如有必要, 请在 "和 #34 中手动编辑出其他 non-nerve 曲面; 编辑和 #34; 通过按住控制键以突出显示多个对象, 然后使用 Delete 键删除它们. </li>
	</ol> </li> <li> 隔离神经特定的荧光线粒体信号: <ol> <li> 选择神经表面的 "编辑" 选项卡以查看和 #34; 掩码属性和 #34; 功能。3.3 步所产生的神经表面被用来隔离那些神经内的线粒体, 远离与角质形成细胞相关的线粒体信号. </li>
		<li> #34; 掩码 #39; 按钮打开 #34; 掩码通道和 #34; 窗口, 从下拉菜单中选择积的红色荧光信号; 通道选择和 #34; 用于线粒体信号. </li>
		<li> 在应用掩码和 #34 之前, 在框中单击以在 #34;d 重复通道中放置一个复选标记; 选项. </li>
		<li> 单击 #34 前面的单选按钮; 常量内/外/#34; 掩码设置的选项, 并在框中单击以在和 #34 中放置复选标记; 将像素设置为外部曲面和 #34; 选项和键入值0.00。单击 OK 按钮创建新的通道, 表示在神经表面的线粒体信号. </li>
	</ol> </li> <li> 创建特定于线粒体的曲面: <ol> <li> 使用和 #34; 创建曲面和 #34; 工具, 使一个固体表面的线粒体从新创建的荧光通道的神经特异性线粒体信号从步骤 3.4. </li>
		<li> 取消选中和 #34; 平滑和 #34; 特征并选择 #34; 背景减法和 #34; 功能来设置阈值。该功能利用线粒体信号周围的局部对比来识别背景中的线粒体. </li>
		<li> 设置阈值足够低, 以准确识别线粒体。在本例中, 较低的阈值为16位 (65536) 的刻度设置为 2000. </li>
		<li> 根据大小筛选线粒体表面。在这个例子中, 体素限制被设置为1.0 体, 这是最低限制可能。如有必要, 手动编辑和 #34 中的 non-mitochondria 曲面; 编辑和 #34; 通过按住控制键以选择多个对象, 然后使用 Delete 键删除它们. 
		注: 有时, 该软件将创建与可分辨的荧光线粒体信号不相关的表面。在这些情况下, 可以使用 and #34 删除它们; 编辑和 #34; 选项卡. </li>
	</ol> </li> <li> 导出并计算分析的形态学值: <ol> <li> 从和 #34 中导出神经和线粒体表面的值; 统计和 #34; 使用电子表格软件进行进一步分析的选项卡. </li>
		<li> 导出神经和线粒体表面的音量值. </li>
		<li> 计算每个图像中存在的单个神经纤维的数量, 并在电子表格中记录该值. </li>
		<li> 对于每个图像, 在电子表格中计算以下潜在的分析值: <ol> <li> 总和所有神经表面的体积. </li>
			<li> 将特定于神经的线粒体表面过滤出小于0.02 和 #181 的体积; m 3 , 并按大小对曲面进行装箱。例如, 使用垃圾箱, 如 0.02-0.04, 0.04-0.08, 0.08-0.16, 0.16-0.32, 0.32-0.64, 0.64-1.28, 1.28-2.56, 大于2.56 和 #181; m 3 . 
			注意: 线粒体容量的下限设置为0.02 和 #181; m 3 基于以前发布的卷 mitonchodria 36 、 37 、 38 . </li>
			<li> 计算每个 bin 中特定神经的线粒体表面的数量, 以及在垃圾箱 (0.02-大于2.56 和 #181; m 3 ) 上的总曲面数. </li>
			<li> 计算每个纸盒中特定神经的线粒体表面的比例。使用每个 bin 的计数除以线粒体表面的总数量. </li>
			<li> 总和所有神经特定的线粒体表面的体积. </li>
			<li> 通过将总神经面积除以所有线粒体表面体积的总和来计算线粒体信号的神经比例. </li>
			<li> 通过将总神经表面体积除以所有线粒体表面的计数来计算每个神经体积的线粒体数. </li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

没有利益冲突要申报。

本议定书的目的是分离, 量化和分析的大小和分布的神经特异性线粒体在 IENFs 3D 从人体皮肤活检。协议中有几个关键步骤。自由浮动荧光免疫组织化学设计用于对每个样本中的多个信号进行着色和分析, 为探索性研究提供了一种更加通用的方法44,45。这一过程允许抗体渗入组织, 以最大限度地获得图像整个50µm 部分, 这是必要的获得共聚焦显微镜图像和3D ...

线粒体提供重要的细胞功能, 包括产生细胞能量, 缓冲钙, 调节坏死和凋亡细胞死亡1,2,3。神经系统具有很高的新陈代谢率, 与身体的4相比, 这表明神经元通过线粒体呼吸以三磷酸腺苷 (ATP) 的形式产生高度的细胞能量。许多证据证明, 神经元函数依赖于 ATP5, 特别是在突触的6。因此, 神经元内线粒体的分布是很重要的。
在过去的10年里, 大量的信息表明, 神经元线粒体的贩运和对接受到高度的管制。运动蛋白参与在神经元中分布线粒体到特定的细胞隔间。线粒体的贩运是特别重要的, 因为神经元项目轴突和树突远离躯体。驱动马达蛋白质主要直接顺 (远离体细胞) 贩运的线粒体沿微管, 而蛋白马达蛋白直接逆行 (向 soma) 运动7,8,9,10. 有细胞信号, 这样的线粒体膜电位和冲动传导, 影响线粒体贩运的存在和方向11,12,13。
除了传送线粒体外, 还有专门的蛋白质将线粒体定位到具有高能量需求的特定蜂窝室, 如郎和突触的节点8,14,17. 实际上, 轴突内的大部分线粒体是 non-motile9、13、18。专门的蛋白质像 syntaphilin 锚定线粒体的微管沿轴突, 而其他蛋白质锚定线粒体的肌动蛋白细胞骨架19-21。生长因子和钙等离子被报告支持停止线粒体运动, 将它们本地化为需要的区域21,22,23。
两者结合在一起, 线粒体的贩运和对接对神经元的正常功能至关重要。为支持这一点, 线粒体贩运的中断与包括阿尔茨海默病、肌萎缩侧索硬化症、腓骨-玛丽牙病、亨廷顿氏病、遗传性痉挛截瘫, 和视神经萎缩15,24,25,26,27。最近的研究侧重于线粒体功能障碍和病理作为糖尿病神经病变的潜在机制, 与糖尿病相关的感觉丧失28,29,30,31 ,32,33。假设是糖尿病改变了皮肤神经末端感官投射中线粒体的分布。因此, 在 intraepidermal 神经纤维 (IENFs), 背根神经节感觉传入的远端提示中, 开发了一种技术来可视化和量化线粒体。该技术将特定线粒体和神经纤维标签的荧光免疫组化与共焦显微镜相结合, 用强大的3D 图像分析软件对信号进行 z 系列采集, 测量神经特异性的分布线粒体从人体皮肤穿孔活检达到这个目的。

<table><tbody><tr><td>2% Zamboni&#039;s Fixative</td><td>Newcomer Supply, Middleton, WI&amp;nbsp;</td><td>1459A</td><td>2% paraformaldehyde, 0.2% saturated picric acid in phosphate buffered saline (PBS), pH 7.4</td></tr><tr><td>10x Phosphate Buffered Saline (PBS)&amp;nbsp;</td><td>Fisher Scientific, Pittsburgh, PA</td><td>BP399-4</td><td>To make up 1x PBS</td></tr><tr><td>Image-iT FX Signal Enhancer</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>I36933</td><td>enhances Alexa Fluor dye signals by reducing nonspecific binding</td></tr><tr><td>Anti-Protein Gene Product 9.5 Antibody (Rabbit Polyclonal)</td><td>Proteintech Group Inc. Rosemont, IL</td><td>14730-1-AP</td><td>abbreviated as PGP9.5, replaces discontinued AbD Serotec (Cat. No. 7863-0504) antibody</td></tr><tr><td>Anti-Pyruvate Dehydrogenase E2/E3bp Antibody (Mouse Monoclonal)</td><td>abcam, Cambridge, MA</td><td>ab110333</td><td>abbreviated as PDH</td></tr><tr><td>Goat anti-mouse Secondary antibody Alexa Fluor 594 conjugate</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>A-11034</td><td>red-fluorescent conjugated secondaryantibody</td></tr><tr><td>Goat anti-rabbit Secondary antibody Alexa Fluor 488 conjugate</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>A-11032</td><td>green-fluorescent conjugated secondaryantibody</td></tr><tr><td>Albumin, from Bovine Serum</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A7906-100</td><td>abbreviated as BSA</td></tr><tr><td>Triton X- 100</td><td>Sigma-Aldrich, St. Louis, MO</td><td>T9284</td><td>abbreviated as TX-100</td></tr><tr><td>0.22 &amp;micro;m Filter</td><td>EMD Millipore, Billerica&lt;br/&gt; MA</td><td>MILLEX GP SLGP 033NS</td><td>0.22 &amp;micro;m Millipore filter</td></tr><tr><td>Parafilm M</td><td>Fisher Scientific, Pittsburgh, PA</td><td>13-374-10</td><td>Curwood Wisconsin LLC Parafilm M (PM-996)</td></tr><tr><td>Non-calibrated Loop</td><td>Fisher Scientific, Pittsburgh, PA</td><td>22-032092</td><td>inoculating Loop by Decon LeLoop (MP 199-25)</td></tr><tr><td>96-well Assay Plate</td><td>Corning Incorporated, Corning, NY</td><td>3603</td><td>96-well flat bottom plate</td></tr><tr><td>Prolong Gold antifade reagent with DAPI</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>P-36931</td><td>DAPI staining of nuclei</td></tr><tr><td>Microscope Cover Glass 50 x 24 mm</td><td>Fisher Scientific, Pittsburgh, PA</td><td>12-544E</td><td>Coverslips</td></tr><tr><td>Superfrost Plus Microscope Slides</td><td>Fisher Scientific, Pittsburgh, PA</td><td>12-550-15</td><td>Microscope Slides</td></tr><tr><td>Leica SP5 Laser Scanning Confocal Microscope</td><td>Leica Microsystems, Buffalo Grove, IL</td><td>SP5</td><td>Confocal Microscope</td></tr><tr><td>Volocity x64 Software&amp;nbsp;</td><td>Perkin Elmer, Waltham , MA</td><td>version 4.4.0</td><td>Volocity software is used for Steps 3.1 and 3.2 in the protocol for image processing</td></tr><tr><td>Imaris x64 3 Dimensional Analysis Software</td><td>Bitplane, Concord, MA</td><td>version 7.7.1</td><td>Imaris software is used for Steps 3.3 through 3.5 in the protocol for image analysis</td></tr><tr><td>Excel</td><td>Microsoft, Redmond, WA</td><td>version Office 2013</td><td>Excel spreadsheet software is used for Step 3.6 in the protocol to summarize morphometric features</td></tr><tr><td>Optimum Cutting Temperature Compound</td><td>Sakura Finetek USA, Inc., Torrance, CA</td><td>4583</td><td>abbreviated as OCT</td></tr><tr><td>Leica Cryostat</td><td>Leica Biosystems, Buffalo Grove, IL</td><td>CM1850</td><td>Cryostat for cutting 50 &amp;micro;m sections</td></tr><tr><td>CellLight Mitochondria-GFP, BacMam 2.0</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>C10600</td><td>Used as a postive control to label mitochondria with a green fluorescent signal</td></tr></tbody></table>

three-dimensional imaging and analysis of mitochondria within human intraepidermal nerve fibers

本议定书的目的是研究 intraepidermal 神经纤维内的线粒体。因此, 3D 成像和分析技术的发展, 以隔离神经特异性线粒体和评估疾病诱导的线粒体远端的感觉神经的变化。该协议结合荧光免疫组织化学、共聚焦显微镜和3D 图像分析技术来可视化和量化神经特异性线粒体。详细的参数在整个过程中定义, 以提供一个具体的例子, 如何使用这些技术来隔离神经特异性线粒体。抗体被用来标记的神经和线粒体信号的组织切片的皮肤穿孔活检, 其次是间接免疫荧光可视化的神经和线粒体与绿色和红色的荧光信号分别。利用共焦显微镜对 Z 系列图像进行采集, 并采用3D 分析软件对信号进行处理和分析。没有必要按照内部描述的确切参数, 但重要的是要与整个染色, 获取和分析步骤中选择的一致。该协议的优点是, 它适用于各种情况, 其中一个荧光信号被用来隔离其他的信号, 否则是不可能单独研究。

人 IENFs 中线粒体的可视化与量化
荧光免疫组织化学允许在人体皮肤活检中同时标记多个信号, 以可视化神经、线粒体和细胞核。96井板是组织免疫组化过程中的步骤的一种简便方法。图 1显示此配置占了多达8节, 可通过12阶段的解决方案进行处理。自由浮动的方法, 加上温和的搅拌与平摇杆, ?...

Watch this Scientific Journal Video about Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers at JoVE.com

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

该协议使用三维 (3D) 成像和分析技术来可视化和量化神经特异性线粒体。这些技术适用于在其他情况下, 一个荧光信号被用来隔离一个子集的数据, 从另一个荧光信号。

人 Intraepidermal 神经纤维内线粒体三维成像及分析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

three-dimensional-imaging-analysis-mitochondria-within-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.2K 次观看.  University of Michigan Medical School. 该协议使用三维 (3D) 成像和分析技术来可视化和量化神经特异性线粒体。这些技术适用于在其他情况下, 一个荧光信号被用来隔离一个子集的数据, 从另一个荧光信号。

视频: Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

三维成像伤害表皮内神经纤维在人体皮肤活检

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

科研

医学

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

神经科学

3D 技术可视化胰腺癌细胞中的线粒体嵴

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

作者聚焦:胰腺癌细胞线粒体超微结构变化可视化的三维技术  

This protocol describes how to reconstruct mitochondrial cristae to achieve 3D imaging with high accuracy, high resolution, and high throughput.

一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a ...

癌症研究

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 ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

使用串行块面部扫描电子显微镜脑线粒体分析

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

切除小鼠神经神经纤维神经丝的成像与分析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

人 Intraepidermal 神经纤维内线粒体三维成像及分析

摘要

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此视频中的章节

三维成像伤害表皮内神经纤维在人体皮肤活检

在皮肤中的单神经末梢体内双光子显微镜

一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

内吞体动力学在生活神经末梢与四维荧光成像可视化

使用串行块面部扫描电子显微镜脑线粒体分析

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

切除小鼠神经神经纤维神经丝的成像与分析

扩展轴突转运体内成像工具包