Name: quantification of subcellular glycogen distribution in skeletal muscle fibers using transmission electron microscopy
Uploaded: 2022-02-07T13:00:00
Description: Watch this Scientific Journal Video about Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy at JoVE.com

这项工作得到了瑞典奥林匹克委员会的支持。

使用人类活检骨骼肌样本的本方案已获得南丹麦卫生研究伦理区域委员会（S-20170198）的批准。在皮下给予局部麻醉（每个切口1-3mL利多卡因2%）后，使用Bergström针从 蚴蚴外侧 肌的皮肤切口进行肌肉活检。如果使用孤立的整只大鼠肌肉，根据丹麦欧登塞大学医院动物伦理委员会的指导方针，在获得肌肉活检之前，通过颈椎脱位处死动物。
1. 原发性固定、后固定、嵌...

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该方法的关键步骤是在固定后使用亚铁氰化钾还原锇。这种修饰的固定剂用于糖原检测的选择性不能用化学完全解释，但也包括实验结果，表明在已知不含糖原的组织中或细胞外空间中没有检测到这种颗粒11。
关键参数是估计值的精度和光纤间的变化。通过遵循本成像方案，获得每根纤维不同糖原池估计值的0.1至0.2之间的误差系数。此误差水平远低于单个光...

糖原颗粒由葡萄糖和各种相关蛋白的支化聚合物1 组成，在高代谢需求期间构成重要燃料2。虽然糖原颗粒尚未得到广泛认可，但它也构成了一种局部燃料，其中一些亚细胞过程优先利用糖原，尽管还有其他更持久的燃料如血浆葡萄糖和脂肪酸3，4。
几篇综述5，6主要基于透射电子显微镜（TEM）7，8对糖原亚细胞分布的一些最早文献，讨论了将糖原储存为亚细胞特异性局部燃料的重要性。第一项研究使用不同的方案来增加糖原从组织化学染色技术到阴性和阳性染色的对比度9，10。一个重要的方法论发展是用亚铁氰化钾还原锇11，12，13，14改进了固定后方案，显著改善了糖原颗粒的对比度。这种改进的方案并未用于一些关于运动诱导的糖原消耗的开创性工作15，但由Graham及其同事重新引入16，17。
基于二维图像，糖原的亚细胞分布最常被描述为位于三个池中的糖原颗粒：构膜下（就在表面膜下），肌原间（肌原纤维之间）或肌原纤维内（肌原纤维内）。然而，糖原颗粒也可以被描述为与例如肌质网7 或细胞核18相关。除了亚细胞分布外，TEM估计的糖原含量的优点还在于可以在单纤维水平上进行定量。这允许研究纤维间的变异性，并对纤维类型和细胞成分（如线粒体和脂滴）进行相关性分析。
这里，描述了TEM估计的骨骼肌纤维中三种常见的糖原亚细胞池（构膜下，肌原间和肌原内）的纤维类型特异性体积含量的方案。该方法已应用于人类19，大鼠20和小鼠21的骨骼肌;以及鸟类和鱼类22;和来自大鼠的心肌细胞23。

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			<td>Philips CM 100 Transmission EM</td>
			<td>Philips</td>
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			<td>Ampliqon.com</td>
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			<td>Ultra-microtome Leica UC7</td>
			<td>Leica</td>
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			<td>Ultrostain lead citrate 3%, stabilised solution</td>
			<td>Leica</td>
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quantification of subcellular glycogen distribution in skeletal muscle fibers using transmission electron microscopy

通过使用透射电子显微镜，可以获得含有单个肌肉纤维的固定样品的高分辨率图像。这样可以量化超微结构方面，例如体积分数，表面积与体积比，形态测量和不同亚细胞结构的物理接触位点。在20世纪70年代，开发了一种用于增强细胞中糖原染色的方案，并为使用透射电子显微镜对糖原和糖原粒度的亚细胞定位的一系列研究铺平了道路。虽然大多数分析将糖原解释为它均匀分布在肌肉纤维内，仅提供单个值（例如，平均浓度），但透射电子显微镜显示糖原作为离散糖原颗粒储存在不同的亚细胞区室中。在这里，描述了从组织收集到定量测定单个骨骼肌纤维的不同亚细胞区室中糖原的体积分数和粒径的分步方案。考虑如何1）收集和染色组织标本，2）执行图像分析和数据处理，3）评估估计的精度，4）区分肌肉纤维类型，以及5）方法学陷阱和局限性。

使用该协议，糖原颗粒看起来是黑色和不同的（图1 和 图2）。糖原的正常值如图 3所示。这些数据基于在不同的先前研究中收集的来自41名健康年轻男性的362种纤维19，24，29，30，31。在这里，可以看出肌原?...

Watch this Scientific Journal Video about Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy at JoVE.com

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

改进的固定后程序增加了组织中糖原颗粒的对比度。本文提供了一个分步方案，描述了如何处理组织，进行成像，并使用立体学方法获得骨骼肌中纤维类型特异性亚细胞糖原分布的无偏和定量数据。

使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

单细胞和亚细胞组织中糖原含量的测量很重要，因为在大多数细胞类型中，单细胞对不断变化的生理条件和疾病的适应方式不同。因此，这种技术的主要优点是透射电子显微镜的分辨率非常高。这提高了具有局部亚细胞结构的可能性，并且在此技术中的这种含量中，糖原染色是无抗体的，这一点很重要。因此，我们没有任何具体问题需要担心。为了获得更好的糖原对比度，只需使用1.5%亚铁氰化钾代替亚铁氰化钾。通过在2毫升微量离心管中0.1摩尔二甲羟丁酸钠缓冲液中加入2.5%戊二醛来制备1.6毫升初级固定溶液。将其储存在5摄氏度下，最多14天。从肌肉活检或整个肌肉中分离出一个小样本，该标本在任何方向上的最大直径为一毫米，并且纵向长于横截面。将试样放入含有冷初级固定溶液的管中。将其储存在5摄氏度下24小时。然后在0.1摩尔二甲酸钠缓冲液中洗涤标本四次。使用转印移液器，从试管中取出用过的缓冲液，使试样保持原样不变，然后加入新鲜的缓冲液。用1%四氧化锇和1.5%亚铁氰化钾在0.1摩尔二甲苯二酸钠缓冲液中以4摄氏度后固定标本120分钟。在室温下用双蒸馏水冲洗试样两次。通过在室温下浸没在一系列分级乙醇中脱水。使用上述体积比在室温下用环氧丙烷和环氧二烯树脂级分级混合物浸润试样。第二天，将试样嵌入模具中100%新鲜的epossidic树脂中，并在60摄氏度下聚合48小时。将标本块安装在超薄切片机支架上。用剃须刀片修剪表面上的块状物以达到组织水平。将金刚石刀安装在样品前面，并将样品表面平行于刀对齐。用金刚石刀制作一微米厚的半薄切片，以检查样品的方向。用甲苯胺蓝染色半薄切片，以便用光学显微镜观察。然后进一步修剪块以减少感兴趣的区域，以获得适当的超薄部分。用第二把金刚石刀切割 60 至 70 纳米厚度的超薄切片。使用完美的循环在一个完整的铜网格上收集一到两个部分。为了对比切片，将网格浸入醋酸铀酰溶液中20分钟，并在双蒸馏水中洗涤网格，然后将网格浸入柠檬酸铅中15分钟，并在双蒸馏水中重新洗涤网格。打开透射电子显微镜计算机和图像记录软件，使用数字慢扫描CCD相机和相关成像软件记录数字图像。在显微镜载物台中插入具有多个切片的网格后，首先以低放大倍率筛选网格，以选择最优质的切片并确定肌肉纤维的方向。下一个焦点，图像在放大倍率高于30， 000时，光束以该部分的外围光纤为中心，并以所需的放大倍率记录一秒钟曝光时间的图像。确保图像以随机的机器人系统顺序分布在光纤的长度和宽度上，以获得无偏的结果并重复成像，直到对6到10根光纤进行成像。通过单击文件，然后单击打开将图像导入图像 J。通过单击“分析”，然后设置比例，将全局比例设置为与图像的原始大小匹配。要放大100%，请单击图像，转到缩放并单击。使用工具菜单中的直线工具测量肌原纤维空间每个图像一个Z盘的厚度，并计算6到10根纤维中每根纤维的平均Z盘厚度。使用分段线工具测量在构体下区域正下方可见的最外层肌原纤维的长度。要插入网格，请单击“分析”，选择工具，然后选择“网格”。现在将每个点的面积设置为32， 400纳米。在 12 个十字击中穹窿下糖原的 12 个附属构象图像中，计算可用长度内的命中次数。通过单击分析插入网格，然后选择工具，然后选择网格并将每个点的面积设置为 160， 000 平方米。计算第 12 个肌原纤维图像中十字击中肌内间隙的命中次数。然后，要插入网格，请单击“分析”，选择工具，然后选择“网格”。现在将每个点的面积设置为3， 600平方米。在第12个肌原纤维图像中，交叉击中肌原的命中次数。通过单击分析插入网格，然后选择工具，然后选择网格并将每个点的面积设置为 32， 400 纳米。在第12个肌原纤维图像中，交叉击中肌原的命中次数。使用32， 400平方米的网格随机选择糖原颗粒，从左上方开始，必要时向左移动。使用直线工具，为12个图像中的每张图像测量每个池中随机选择的五个糖原颗粒的直径，以获得每根光纤每个池的平均60个颗粒。该图显示了三个糖原池的正常值。可以观察到，肌原间糖原值的分布接近正常，而肌原和构肌下糖原均显示出偏斜分布，纤维有时具有过量的糖原。在所有步骤中，使用亚铁氰化钾非常重要。糖原的分析可以与线粒体和脂滴的分析相结合，这可以提高我们对其他关键代谢成分如何与糖原和肌肉健康相关的理解。

Research

JoVE Journal

Biochemistry

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

改进的小鼠骨骼肌染色质沉淀的协议

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analysis of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

使用支撑浮块在透射电子显微镜中制备样品支撑膜

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

微图案透射电子显微镜网格，用于在全细胞冷冻电子断层扫描工作流程中指导细胞定位

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

骨骼肌线粒体蛋白质导入能力的测定

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis FACE Method

使用荧光团辅助碳水化合物电泳（FACE）方法测定糖原的葡聚糖链长分布

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

使用200 KV透射电子显微镜常规收集高分辨率冷冻电镜数据集

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution (Video) | JoVE 

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...