JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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생물학

使用连续截面电子显微镜对肝细胞中组织间接触位点进行三维表征

Published: June 09, 2022
doi:
10.3791/63496
, , ,
1MRC Laboratory for Molecular Cell Biology,University College London, 2NIHR Great Ormond Street Hospital Biomedical Research Centre,University College London

Summary

一种简单而全面的方案，用于获取肝脏肝细胞器或其他组织中细胞之间细胞器之间膜接触位点的三维细节。

Abstract

长期以来，透射电子显微镜一直被认为是细胞超微结构可视化的黄金标准。然而，分析通常局限于二维，妨碍了全面描述细胞器之间三维（3D）超微结构和功能关系的能力。体积电子显微镜（vEM）描述了一系列技术，这些技术能够以中尺度，微尺度和纳米级分辨率在3D中询问细胞超微结构。

该协议提供了一种可访问且可靠的方法，可以使用串行截面传输EM（TEM）采集vEM数据，并在单个简单的工作流程中涵盖样品处理到数字3D重建的技术方面。为了证明该技术的有用性，提出了内质网和线粒体及其在肝细胞中的接触位点之间的3D超微结构关系。组织间接触在细胞器之间的离子、脂质、营养物质和其他小分子的转移中起着至关重要的作用。然而，尽管它们在肝细胞中最初被发现，但关于它们的物理特征，动力学和功能仍然有很多需要了解的地方。

舌间接触可以显示一系列形态，在两个细胞器彼此之间的接近程度（通常约为10-30nm）和接触部位的范围（从点状接触到较大的3D蓄水池样接触）方面有所不同。密切接触者的检查需要高分辨率成像，连续切片TEM非常适合可视化肝细胞分化过程中组织间接触的3D超微结构，以及与代谢疾病相关的肝细胞结构的改变。

Introduction

自20世纪30年代发明以来，电子显微镜使研究人员能够可视化细胞和组织的结构成分1，2。大多数调查都提供了2D信息，因为构建3D模型需要费力的串行截面收集，手动摄影，负片处理，手动描摹以及从玻璃，塑料或聚苯乙烯泡沫塑料3，4板中创建和组装3D模型。近70年后，该过程的许多方面都取得了相当大的进步，从显微镜性能，串行切片收集，自动数字成像，用于3D重建，可视化和分析的复杂软件和硬件到现在统称为体积EM（vEM）的替代方法。这些vEM技术通常被认为在微米尺度上以纳米分辨率提供3D超微结构信息，并包括透射电子显微镜（TEM）和较新的扫描电子显微镜（SEM）技术;请参阅评论5，6，7，8。

例如，聚焦离子束SEM（FIB-SEM）使用SEM内部的聚焦离子束在对块表面进行顺序SEM成像扫描之间铣削掉块的表面，从而允许对样品进行重复的自动铣削/成像，并建立用于重建的3D数据集9，10。相反，串行块面SEM（SBF-SEM）在成像11，12之前使用SEM内部的超薄切片机从块面上除去材料，而阵列断层扫描是一种非破坏性过程，需要收集串行部分，在盖玻片，晶圆或胶带上，然后设置自动成像SEM中连续部分的感兴趣区域以生成3D数据集13.与阵列断层扫描类似，串行截面TEM（ssTEM）要求在成像之前收集物理截面;但是，这些部分被收集在TEM网格上，并在TEM14，15，16中成像。ssTEM可以通过执行倾斜断层扫描17，18，19来扩展。连续倾斜断层扫描在 x， y 和z中提供最佳分辨率 ， 虽然它已被用于重建整个细胞20，但它具有合理的挑战性。该协议侧重于ssTEM的实际方面，作为许多EM实验室最容易获得的vEM技术，这些实验室目前可能无法访问专门的切片或vEM仪器，但可以从生成3D vEM数据中受益。

用于3D重建的连续超显微切除术以前被认为是具有挑战性的。很难切割均匀截面厚度的直带，能够以正确的顺序排列和拾取正确尺寸的丝带，并将它们放在具有足够支撑的网格上，但是没有网格条遮挡感兴趣的区域，最重要的是，在不丢失截面的情况下，因为不完整的系列可能会阻止完整的3D重建21。然而，对商用超微球切割机、金刚石切割和修整刀22，23、网格21，24上的电子光亮支撑膜以及用于辅助截面附着力和色带保存的粘合剂13，21 的改进只是多年来使该技术在许多实验室中更加常规的一些渐进式进步。一旦收集了连续切片，TEM中的串行成像就很简单，并且可以提供具有 x 和y的亚纳米px大小的EM图像 ， 从而允许对亚细胞结构进行高分辨率询问 – 这是许多研究问题的潜在要求。这里介绍的案例研究展示了ssTEM和3D重建在肝细胞内质网（ER）-细胞器接触研究中的使用，其中ER-细胞器接触首次观察到25，26。

在与核包膜连续的同时，ER还与许多其他细胞器密切接触，包括溶酶体，线粒体，脂质液滴和质膜27。ER-细胞器接触与脂质代谢28，磷酸肌醇和钙信号传导29，自噬调节和应激反应30，31有关。ER-细胞器接触和其他器官间接触是高度动态的结构，对细胞代谢需求和细胞外线索作出反应。它们已被证明在形态学上在其大小和形状以及细胞器膜32，33之间的距离上有所不同。据认为，这些超微结构差异可能反映了它们不同的蛋白质/脂质组成和功能34，35。然而，定义组织间接触并对其进行分析仍然是一项具有挑战性的任务36.因此，需要一种可靠而简单的方案来检查和表征组织间接触，以便进一步检查。

由于ER-细胞器接触的范围在膜到膜分离中的范围为10至30nm，因此鉴定的金标准历来是TEM。薄切片TEM已经揭示了驻留ER蛋白在不同膜接触处的特定亚域定位37。传统上，这揭示了具有nm分辨率的ER细胞器接触，但通常仅呈现这些相互作用的2D视图。然而，vEM方法以3D形式揭示了这些接触部位的超微结构呈现和背景，能够完全重建接触点并更准确地对接触物进行分类（点状vs管状vs.蓄水管样）和定量38，39。除了作为第一个观察到ER-细胞器接触的细胞类型25，26之外，肝细胞还具有广泛的其他器官间接触系统，这些系统在其结构和生理学中起着至关重要的作用28，40。然而，肝细胞中 ER 细胞器和其他器官间接触的彻底形态学表征仍然缺乏。因此，在再生和修复过程中，组织间接触如何形成和重塑与肝细胞生物学和肝功能特别相关。

Protocol

所有动物都按照英国内政部的指导方针进行饲养，组织收获是根据1986年英国动物（科学程序）法案进行的。 1. 标本固定和制备 将肝组织解剖成适当大小的碎片，约8 mm x 8 mm x 3 mm，并将切片置于温磷酸盐缓冲盐水（PBS，37°C）中。 将室温（20-25°C）固定剂（1%蔗糖中的1.5%戊二醛，0.1M二甲酸钠）注射到肝脏碎片中，并在室温下将其从PBS转移到固定剂?…

Representative Results

对于这种技术，根据生物学研究目的选择感兴趣的区域，并在修剪和分割嵌入的组织之前确定。同样，块面的大小可能由研究问题决定;在这种情况下，对样品进行修剪以留下约0.3 mm x 0.15 mm的块面（图4A）。这允许每个网格有两个9个序列部分的网格，提供18个串行部分，并包含体积约为62μm3 （316μm x 150μm x 1.3μm）的肝组织体积。该体积足以允许肝脏组织中单个线粒体…

Discussion

该协议描述了一种可访问的vEM技术，用于可视化3D中的细胞器结构和相互作用。肝细胞中组织间接触的形态学作为案例研究在这里提出。然而，这种方法也被应用于研究各种其他样品和研究领域，包括周围神经45中的Schwann细胞 – 内皮相互作用，内皮细胞46中的Weibel Palade体生物发生，肾细胞47中的货物分泌以及海马神经元中的突触形态<sup class="xre…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢Joanna Hanley，Rebecca Fiadeiro和Ania Straatman-Iwanowska的专家技术援助。我们还感谢Stefan实验室成员和Ian J. White的有益讨论。J.J.B由伦敦大学学院MRC分子细胞生物学实验室的MRC资助，奖励代码MC_U12266B。C.J.S.由MRC资助，资助伦敦大学学院分子细胞生物学大学MRC实验室，奖励代码MC_UU_00012/6。P.G.由欧洲研究理事会资助，拨款代码ERC-2013-StG-337057。

Materials

0.22 µm syringe filter Sarstedt 83.1826.001
Aluminum trays Agar Scientific AGG3912
Amira v6 ThermoFisher https://www.thermofisher.com
Chloroform Fisher C/4960/PB08
DDSA/Dodecenyl Succinic Anhydride TAAB T027 Epon ingredient
Diamond knife DiaTOME ultra 45°
DMP-30/2,4,6-tri (Dimethylaminomethyl) phenol TAAB D032 Epon ingredient
Dumont Tweezers N5 Agar Scientific AGT5293
Fiji https://imagej.net/
Fiji TrakEM2 plugin https://imagej.net/
Formaldehyde 36% solution TAAB F003
Formvar coated slot grid Homemade Alternative: EMS diasum (FF2010-Cu)
Glass bottle with applicator rod Medisca 6258
Glass vials Fisher Scientific 15364769
Gluteraldehyde 25% solution TAAB G011
MNA/Methyl Nadic Anhydride TAAB M011 Epon ingredient
Osmium Tetroxide 2% solution TAAB O005
Potassium Ferricyanide Sigma-Aldrich P-8131
Propylene oxide Fisher Scientific E/0050/PB08
Reuseable adhesive Blue Tack
Reynolds Lead Citrate TAAB L037 Section stain
Sodium Cacodylate Sigma-Aldrich C-0250 to make 0.1 M Caco buffer
Super Glue RS Components 918-6872 Cyanoacrylate glue, Step 1.3
TAAB 812 Resin TAAB T023 Epon ingredient
Tannic acid TAAB T046
Triton X-100 Sigma-Aldrich T9284
Two part Epoxy Resin RS Components 132-605 Alternative: Step 2.13
Ultramicrotome Leica UC7
Vibrating microtome Leica 100 µm thick slices, 0.16 mm/s cutting at 1 mm amplitude .
Weldwood Original Contact cement DAP 107 Contact adhesive: Step 3.1.4
DOWNLOAD MATERIALS LIST

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Keywords: 3D ReconstructionInterorganelle Contact SitesHepatocytesSerial Section Electron MicroscopyOrganelle MorphologyCellular TraffickingUltra-thin SectioningMicrotomeDiamond KnifeCutting WindowRibbon SectioningSlot Grid
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Chun Chung, G. H., Gissen, P., Stefan, C. J., Burden, J. J. Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy. J. Vis. Exp. (184), e63496, doi:10.3791/63496 (2022).
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