这项研究是在细胞和有机体生物学研究所和台湾台北中央研究院的低温 em 核心进行的。这项工作得到了中央研究院和大多数的支持。

1. 使用振动叶片切片的切片果蝇组织
<ol>
	<li>麻醉果蝇在冰上, 将每只苍蝇浸泡在1毫升的4% 低熔点琼脂糖的磷酸盐缓冲液中。允许琼脂糖凝固在冰上。通常, 4-6 只苍蝇被处理。</li>
	<li>使用振动刀片切片的部分琼脂糖凝胶嵌入果蝇到切片100µm 厚度和浸泡在定影液中含有2.5% 戊二醛在0.1 米磷酸缓冲液。 注: Vibratome 切片是首选, 因为组织结构保持更完整的比其他方法。另外, 解剖镊子可用于解剖 IFM 的固定液中含有2.5% 戊二醛在0.1 米磷酸缓冲液。</li>
</ol>
2. 用高压冷冻和冷冻置换 (HPF/FS) 法制备 EM 试样
<ol>
	<li>在磷酸盐缓冲液的3滴 (150 µL) 中洗涤组织切片, 其次是2滴 (100 µL) 的磷酸盐缓冲液和 20% BSA。然后在金载体中放置部分, HPF 填充有缓冲液和 20% BSA。</li>
	<li>根据用户手册将载有携带者的样品装入高压冷藏箱。</li>
	<li>冷冻后, 在液氮下释放托架, 并将其转移到冷冻替代装置, 冷却到-140 ° c。</li>
	<li>执行表 1中所示的冻结替代协议, FS 鸡尾酒含有2% 戊二醛、2% 锇氧化和0.1% 铀丙酮乙酸酯。</li>
	<li>小心地从携带针的载体上取出标本, 并在室温下将试样嵌入树脂中。聚合树脂在65° c 为16小时。 注意: 应修改 FS 协议以准备其他类型的示例。HPF/FS 是保存超微结构和减少细胞含量损失的首选。<ol>
		<li>或者, 应用化学固定协议。用2.5% 戊二醛固定标本过夜, 用缓冲器冲洗, 然后用1% 锇氧化 2 h. 洗涤和脱水与上升的乙醇浓度, 然后渗透和嵌入在 Spurr 的树脂在65° c 聚合前16h</li>
	</ol>
	</li>
</ol>
3. 制备电子断层扫描标本的系列切片
<ol>
	<li>修剪标本块, 露出所需的块面, 其中含有组织。</li>
	<li>预处理金颗粒 (直径 10 nm) 与 1% BSA, 30 分钟洗涤和悬浮在 PBS 缓冲区中的金粒子。覆盖在铜槽网格上涂上碳膜的金颗粒, 以创建基准标记。</li>
	<li>在透射电镜下检查, 以获得足够的基准标记 (至少 5-10 标记) 在层析成像的视野。</li>
	<li>切割串行部分, 200-250 nm 的厚度, 使用 ultramicrotome。</li>
	<li>在插槽网格上使用一个完美的环形截面来收集串行部分。</li>
	<li>染色的部分与雷诺的铅柠檬酸10分钟。</li>
	<li>在剖面顶部覆盖第二层的基准金粒子。</li>
</ol>
4. 收集双倾斜电子断层扫描
<ol>
	<li>将网格加载到双轴层析成像支架上, 并插入到 200 kV 的透射电子显微镜中。</li>
	<li>将显微镜对准 eucentric 焦点。</li>
	<li>设置自动数据收集软件。调整和对齐电子束在多尺度成像设置。有关操作详细信息, 请参阅用户手册10 (图 2)。</li>
	<li>获取相机黑暗和明亮的参考在一个空白的区域没有碳膜下的断层扫描收集设置。</li>
	<li>在低放大率下收集栅格地图集。选择序列切片上的线粒体作为断层扫描收集的目标。</li>
	<li>获取一个倾斜系列, 从-60 °到 + 60 °, 每个目标的轴上2递增。 注意: 倾斜角度将被机械约束的标本持有人的设计。支架将在高倾角下阻挡光束。</li>
	<li>要收集第二个倾斜系列, 请旋转试样托架90°。获得一个新的地图集。选择相应的位置, 并获取每个目标轴-b 上的倾斜系列。 注: 其他软件包 (如 SerialEM 和 Xplore3D) 可用于自动数据收集。</li>
</ol>
5. 使用软件重构3D 断层和分段子卷
<ol>
	<li>使用 IMOD 软件11重构双轴倾斜图像的断层。
	<ol>
		<li>有关操作细节, 请参阅用户手册。</li>
		<li>将单个倾斜级数与黄金基准标记的位置对齐。通过后向投影方法或由轴 a 和轴 B 的同时迭代重建技术 (SIRT) 方法 (图 3) 重新构造断层。</li>
		<li>结合轴 a 和轴 B 的断层, 生成 double-tilt 图, 减少缺少楔形工件。</li>
		<li>联合 double-tilt 断层的序列部分, 以获得覆盖整个线粒体体积的重建。通过 IMOD 程序对串行段之间的间隙进行建模。</li>
		<li>将连接的断层和纸盒修剪为所需的卷分割大小。</li>
	</ol>
	</li>
	<li>使用3D 软件分析连接的串行断层。
	<ol>
		<li>有关操作细节, 请参阅用户手册。</li>
		<li>用高斯滤镜 (或其他所需方法) 过滤断层, 以改善特征对比度, 降低背景密度。</li>
		<li>对线粒体的超微结构进行人工或自动分割。</li>
		<li>显示细分模型以允许在3D 中进行检查。</li>
		<li>使用3D 中可用的工具生成影片。</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Dahl, R., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+freezing+for+the+preservation+of+biological+structure:+theory+and+practice.">High-pressure freezing for the preservation of biological structure: theory and practice.</a> J Electron Microsc Tech. 13 (3), 165-174 (1989).</li><li>Sabatini, D. D., Bensch, K., Barrnett, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemistry+and+electron+microscopy.+The+preservation+of+cellular+ultrastructure+and+enzymatic+activity+by+aldehyde+fixation.">Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation.</a> J Cell Biol. 17, 19-58 (1963).</li><li>Rigort, A., 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=Micromachining+tools+and+correlative+approaches+for+cellular+cryo-electron+tomography.">Micromachining tools and correlative approaches for cellular cryo-electron tomography.</a> J Struct Biol. 172 (2), 169-179 (2010).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> PLoS Biol. 2 (11), e329 (2004).</li><li>Lucic, V., Forster, F., Baumeister, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+studies+by+electron+tomography:+from+cells+to+molecules.">Structural studies by electron tomography: from cells to molecules.</a> Annu Rev Biochem. 74, 833-865 (2005).</li><li>Soto, G. 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=Serial+section+electron+tomography:+a+method+for+three-dimensional+reconstruction+of+large+structures.">Serial section electron tomography: a method for three-dimensional reconstruction of large structures.</a> Neuroimage. 1 (3), 230-243 (1994).</li><li>Jiang, Y. F., 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=Electron+tomographic+analysis+reveals+ultrastructural+features+of+mitochondrial+cristae+architecture+which+reflect+energetic+state+and+aging.">Electron tomographic analysis reveals ultrastructural features of mitochondrial cristae architecture which reflect energetic state and aging.</a> SciRep. 7, 45474 (2017).</li><li>Cogliati, S., Enriquez, J. A., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Cristae:+Where+Beauty+Meets+Functionality.">Mitochondrial Cristae: Where Beauty Meets Functionality.</a> TrendsBiochemSci. 41 (3), 261-273 (2016).</li><li>Friedman, J. R., Nunnari, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+form+and+function.">Mitochondrial form and function.</a> Nature. 505 (7483), 335-343 (2014).</li><li>Suloway, C., 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=Fully+automated,+sequential+tilt-series+acquisition+with+Leginon.">Fully automated, sequential tilt-series acquisition with Leginon.</a> J Struct Biol. 167 (1), 11-18 (2009).</li><li>Mastronarde, D. N., Held, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+tilt+series+alignment+and+tomographic+reconstruction+in+IMOD.">Automated tilt series alignment and tomographic reconstruction in IMOD.</a> J Struct Biol. 197 (2), 102-113 (2017).</li></ol>

作者声明他们没有竞争的金融利益。

在本协议中, 我们描述了一个优化的工作流程, 应用序列切片电子断层扫描研究的3D 线粒体超微结构的果蝇间接飞行肌肉。在样品中保存超微结构是这类分析的主要技术挑战。为了更好地保存超微结构, 包括两个方法步骤。首先, 用振动叶片切片切片对组织进行取样, 以尽可能地保持组织结构。其次, 对 HPF/FS 协议进行了优化, 以保留细胞的超微结构, 在准备嵌入标本块。试样在高压下被冻结, 降低了水的冰点, 减少了冰晶的形成, 破坏了超微结构的1。厚度为0.1 毫米的试样可以瞬间被玻璃化, 然后进行冷冻置换, 生成标本块进行 EM 分析。与化学固定方法相比, HPF/FS 对超微结构的保存有明显改善。利用这一系列步骤进行样品制备, 线粒体双层膜和嵴膜的保存得到了明显的改善。
获取试样的连续截面是该方法最具挑战性的步骤。由于电子束的穿透力有限, 因此该截面的厚度分别限制为 250 nm 或 500 nm, 使用 TEM 运行在200伏或300伏。由于线粒体的厚度可能超过2µm, 因此需要连续的部分才能获得完整的体积重建。但是, 恢复足够数量的串行部分来跨越整个细胞是一个技术难题。在梯形的基座之间尽可能平整地修剪块面, 可以使插槽网格容纳更多的截面, 从而覆盖较大的卷。此外, 对于精简部分, 使用一个完美的循环会增加将串行部分转移到网格的成功率。
串联电子断层扫描可以用标准的 EM 核心设备来完成。然而, 由于技术的限制, 该方法有一些不可避免的局限性。一是材料不可避免地在连续的区段之间丢失, 在联接的重建留下空白。第二是缺少的楔形工件, 这是由于有限的倾斜角度, 是可以实现的。这种限制发生的原因是, 试样持有者不能完全旋转而不堵塞电子束。尽管有这些限制, 连续切片电子断层扫描提供了足够的分辨率, 以揭示细胞和器官的超微结构在3D。
对于较小规模的成像, 低温电子断层扫描是一种新兴的技术, 可用于获得结构的大分子复合体和程序集原位在 nm 或 sub-angstrom 分辨率与 sub-tomographic 结合重建3。在这种应用中, 细胞通过切片或聚焦离子束在液氮下铣削而变薄。断层是在低温条件下收集的, 在这种情况下, 分子结构保存在靠近原生状态的地方, 没有化学固定、脱水或嵌入。在天平的另一端, 以牺牲分辨率为代价来分析大的组织体积, 串口扫描电子显微镜是一种吸引人的方式, 尽管它需要特定的仪器4。

电子显微镜是一个有价值的工具, 研究的结构上下文的亚细胞组装和设备, 执行蜂窝过程。方法已经开发, 以保持组织或细胞的超微结构, 无论是通过化学固定醛或高压冷冻 (HPF) 后, 冷冻置换 (FS)1,2。嵌入的标本块, 然后可以切片, 染色, 并观察用透射电子显微镜 (TEM)。HPF 标本也可以在低温条件下处理, 如冷冻切片或聚焦离子束 (纤维蛋白原) 铣削, 并通过低温 EM3,4观察。
虽然薄片 EM 提供了丰富的形态学洞察力, 结果2D 图像只能揭示特定截面的超微结构。在3D 体积中组织超微结构的方法仍然模糊不清。为了使三维的细胞超微结构可视化, 在获得了一系列倾斜图像并将其投影到生成层析重建5 (图 1) 的情况下, 开发了一种电子断层扫描方法。双倾斜系列可通过旋转样品90°并获得第二倾斜系列来收集。这将减少因取样角度有限而导致的缺少楔形工件, 并提高图的分辨率。
在这里, 我们描述了用于研究果蝇间接飞行肌 (IFM)6、7、8、9的连续切片电子断层扫描的应用。.为了获得覆盖整个线粒体 (大约2.5 µm 厚) 的3D 重建, 序列切片从果蝇IFM 组织块获得。每个部分的断层使用自动数据收集软件单独收集。层析重建和串行断层与 IMOD 包, 以获得重建体积的整个线粒体。通过3D 软件对加入的断层进行了分析。对线粒体嵴的密度进行了细分, 产生了一个在三维度中揭示组织的分割模型。

<table width="852">
	<tbody>
		<tr height="40">
			<td height="40" style="width:252px;height:40px;">vibrating blade microtome</td>
			<td style="width:123px;">Leica&#160;</td>
			<td style="width:267px;">VT1200S</td>
			<td style="width:212px;">Tissue sectioning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">high-pressure freezer</td>
			<td>Leica&#160;</td>
			<td>EM HPM100</td>
			<td>Specimen preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">freeze-substitution device</td>
			<td>Leica&#160;</td>
			<td>EM AFS2</td>
			<td>Specimen preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ultramicrotome</td>
			<td>Leica&#160;</td>
			<td>EM UC7</td>
			<td>Ultra-thin sectioning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">dual-axis tomography holder</td>
			<td>Fischione&#160;</td>
			<td>Model 2040</td>
			<td>tomography collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">transmission electron microscope</td>
			<td>FEI</td>
			<td>&#160;Tecnai F20</td>
			<td>tomography collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CCD</td>
			<td>Gatan&#160;</td>
			<td>UltraScan 1000</td>
			<td>tomography collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Leginon</td>
			<td colspan="2" style="width:388px;">NRAMM/AMI</td>
			<td>tomography collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IMOD</td>
			<td colspan="2" style="width:388px;">Boulder Laboratory for 3-D Electron Microscopy of Cells</td>
			<td>Tomography reconstruction&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Avizo 3D</td>
			<td>FEI</td>
			<td></td>
			<td>Tomography analysis</td>
		</tr>
	</tbody>
</table>

3d mitochondrial ultrastructure of drosophila indirect flight muscle revealed by serial-section electron tomography

线粒体是细胞的动力, 产生 ATP, 脂质和代谢物, 以及调节钙稳态和细胞死亡。独特的嵴丰富的双膜超微结构的这个细胞是优雅的安排, 以执行多种功能的划分生物分子。线粒体超微结构与各种功能密切相关;然而, 这些结构-函数关系的细微细节才刚刚开始被描述。在这里, 我们展示了在果蝇间接飞行肌肉中应用的序列切片电子断层扫描来阐明线粒体结构。串联电子断层扫描可用于研究三维的任何细胞结构。

应用序列切片电子断层扫描技术分析线粒体嵴的结构特征, 反映其能量状态和衰老。我们发现,果蝇IFM 的线粒体在 3D (图 4)7中形成一个集成的嵴和矩阵网络。此外, 突变蝇与线粒体 DNA 复制缺陷和加速衰老表型积累线粒体, 其中包含洋葱状旋转核心 (图 5)7的分部。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56567/56567fig1.jpg"> 
图 1: 从倾斜级数中重建电子断层的图示.在实验中, 当物体倾斜到不同程度时, 电子束通过3D 物体传递。对于每个倾斜条件, 将生成一个2D 投影并用照相机捕获。然后将2D 投影投影到基于中心切片定理的3D 对象重建中。在插图中, 对于在不同倾角下投射到单个尺寸中的原始2D 图像, 将显示相同的过程。然后使用1D 投影重建2D 图像。<a href="//ecsource.jove.com/files/ftp_upload/56567/56567fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56567/56567fig2.jpg"> 
图 2: 自动数据收集一个图像查看器显示的软件显示的地图集的一个槽网包含串行部分, 多尺度目标的线粒体和获得倾斜图像。缩放栏 = 500 µm (左上面板), 100 µm (左下面板), 1 µm (右面板)。<a href="//ecsource.jove.com/files/ftp_upload/56567/56567fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56567/56567fig3.jpg"> 
图 3: 在果蝇中的单个线粒体的序列剖面电子断层扫描间接飞行肌肉。() 2D 显微和 (B) 3D 断层从覆盖整个线粒体体积的串行部分。(C) 连接的串行节断层被投影以创建纵向剖面, z-axis 垂直显示。值得注意的是, 组织切片导致材料丢失, 在连接的断层之间留下间隙。缩放条形图 = 500 nm。<a href="//ecsource.jove.com/files/ftp_upload/56567/56567fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56567/56567fig4.jpg"> 
图 4: 3D 中显示的集成 intra-mitochondria 嵴和矩阵网络.(A) 线粒体电子断层重建切片, 显示通过 z-axis 的层状膜之间的开关。(B) 右手和/或 left-handed 螺旋的观察到的嵴开关模式的图示。(C) 层析分割, 在 3D (D) 中说明了 left-handed 螺旋的嵴开关模式, 并对分割模型 (E, F) 渲染了颜色。层析切片显示横向基质 70-100 (暗密度, 标记红色) 横跨嵴膜 (白色密度)。(G) 图的分割模型 (E) 显示横向矩阵 70-100 (以红色表示) 和具有代表性的嵴 (灰色)。标尺 = 50 毫微米 (A), 200 毫微米 (C) 和300毫微米 (D, E, F, G)。图为转载于江et al.7 <a href="//ecsource.jove.com/files/ftp_upload/56567/56567fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56567/56567fig5.jpg"> 
图 5: 在老化期间线粒体 DNA 复制缺陷的果蝇中积累的具有洋葱状旋转芯的线粒体.具有代表性的层析切片和相应的分割 (右面板) 显示一个横截面视图 (顶部) 和纵向剖面视图 (底部) 的漩涡核心。通过任意颜色渲染来表示体积分割, 以突出旋涡的核心。缩放栏: 200 nm。图为转载于江et al.7 <a href="//ecsource.jove.com/files/ftp_upload/56567/56567fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>步</td>
			<td colspan="2">临时</td>
			<td colspan="2">时间</td>
			<td colspan="2">解决</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="2">-140 ° c 到-9 ° c</td>
			<td colspan="2">30分钟</td>
			<td colspan="2">液氮</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="2">-90 ° c</td>
			<td colspan="2">96小时</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="2">-90 ° c 到-60 ° c</td>
			<td colspan="2">6小时 (5 ° c/小时)</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="2">-60 ° c</td>
			<td colspan="2">12小时</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="2">-60 ° c 到-25 ° c</td>
			<td colspan="2">7小时 (5 ° c/小时)</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>6</td>
			<td colspan="2">-25 ° c</td>
			<td colspan="2">12小时</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="2">-25 ° c 到0° c</td>
			<td colspan="2">5小时 (5 ° c/小时)</td>
			<td colspan="2">FS 鸡尾酒</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="2">0° c</td>
			<td colspan="2">1小时 x 3 次</td>
			<td colspan="2">丙酮</td>
		</tr>
		<tr>
			<td>9</td>
			<td colspan="2">房间温度</td>
			<td colspan="2"></td>
			<td colspan="2">树脂渗入</td>
		</tr>
	</tbody>
</table>
表 1: 冻结替代议定书。

Watch this Scientific Journal Video about 3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography at JoVE.com

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

连续切片电子断层扫描显示的 3D果蝇间接飞行肌的线粒体超微结构研究

在本协议中, 我们展示了在果蝇间接飞行肌肉中应用的序列切片电子断层扫描来阐明线粒体结构。

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique ...

3d-mitochondrial-ultrastructure-drosophila-indirect-flight-muscle

Research

JoVE Journal

Biology

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

8.4K 次观看.  National Taiwan University. 在本协议中, 我们展示了在果蝇间接飞行肌肉中应用的序列切片电子断层扫描来阐明线粒体结构。

视频: 3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

在果蝇的飞行过程中可视介导的气味追踪

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

科研

生物学

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

果蝇成人间接飞行肌的光学横截肌面积测定

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

通过冷冻软X射线断层扫描对细胞进行3D制图描述

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

优化低温电子断层扫描数据采集策略

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...