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一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

Published: June 23, 2023
doi:
10.3791/65290
, , , , , ,
1School of Pharmaceutical Science of Zhejiang Chinese Medical University, 2Department of Pharmacy,Zhejiang Provincial Dermatology Hospital, 3Academy of Chinese Medical Sciences of Zhejiang Chinese Medical University

Summary

该协议描述了如何重建线粒体嵴以实现高精度，高分辨率和高通量的3D成像。

Abstract

了解细胞器超微结构的动态特征，不仅具有丰富的未知信息，而且从三维（3D）角度来看也很复杂，对于机制研究至关重要。电子显微镜（EM）具有良好的成像深度，并允许重建高分辨率图像堆栈，以研究细胞器的超微结构形态，即使在纳米尺度上也是如此;因此，3D重建因其无与伦比的优势而变得越来越重要。扫描电子显微镜（SEM）提供了一种高通量图像采集技术，允许在连续切片中从相同的感兴趣区域重建3D大型结构。因此，SEM在大规模三维重建中的应用，以恢复细胞器的真实三维超微结构变得越来越普遍。在该协议中，我们建议结合连续超薄切片和3D重建技术来研究胰腺癌细胞中的线粒体嵴。该协议以逐步的方式描述了如何执行这些技术的详细信息，包括锇 – 硫代碳酰肼 – 锇（OTO）方法，连续超薄切片成像和可视化显示。

Introduction

线粒体是细胞中最重要的细胞器之一。它们是细胞生物能量学和代谢的中心枢纽1,2，并在癌症中起着关键作用3。胰腺癌（PC）由于其快速扩散和高死亡率，是最难治疗的癌症之一4。线粒体功能障碍主要由线粒体形态变化3,5,6,7引起，与PC8的疾病机制有关。线粒体也是高度动态的，这反映在其网络连接和嵴结构的频繁和动态变化上9。嵴结构的重塑可直接影响线粒体功能和细胞状态10,11，在肿瘤细胞生长、转移和肿瘤微环境变化过程中发生显著改变12,13。

近年来，科学家们使用EM观察研究了这种细胞器14,15,16,17;例如，研究人员使用3D重建技术分析了线粒体动力学6,7,18,19。电子显微镜图像三维重建的一般概念和方法早在196820年就正式确立，涉及结合电子显微镜、电子衍射和计算机图像处理来重建T4噬菌体尾巴。到目前为止，电子显微镜3D成像技术在图像分辨率21、自动化程度22和处理体积23方面取得了重大进展，并且在生物学研究中的应用越来越广泛，从组织水平到纳米尺度的细胞器超微结构水平24。近年来，电子显微镜3D成像也成为一种前景广阔的技术25,26,27。

对线粒体嵴的日益关注特别说明了超微结构体积成像的基本要求。透射电子显微镜（TEM）已被用于可视化在铜网格（400目）28上收集的样品，电子束穿过该切片。然而，由于铜网格的范围有限，不可能对同一样品29的连续切片进行完全成像。这使得TEM成像过程中靶标结构的研究变得复杂。此外，TEM依赖于耗时且容易出错的手动任务，包括切割和收集多个切片并按顺序成像21，因此它不适用于大体积样品的超微结构重建23。目前，大体积样品成像的高分辨率重建是通过使用专用设备实现的，例如TEM相机阵列（TEMCA）30 或两个第二代TEMCA系统（TEMCA2）31，可以在短时间内实现自动化高通量成像。然而，由于需要定制设备，这种类型的成像不具有易于获得和通用的优点。

与TEM相比，基于SEM 32,33自动生成大面积连续体积图像的方法提高了串行成像的效率和可靠性，并提供了更高的z分辨率34。例如，连续块面扫描电子显微镜（SBF-SEM）和聚焦离子束扫描电子显微镜（FIB-SEM）都使高速，高效和分辨率35,36的超微结构的3D重建成为可能。然而，不可避免地，通过SBF-SEM的金刚石刀或通过FIB-SEM33,37的聚焦离子束进行铣削，块表面被机械剃掉。由于两种方法对样品的破坏性，不可能再次重建相同的目标结构以进行进一步分析38,39,40。此外，很少有研究试图使用EM重建癌细胞的3D细胞器超微结构来观察病理变化12。基于这些原因，为了进一步阐明胰腺癌细胞等癌细胞的病理机制，我们提出了一种使用超薄切片机和场发射扫描电子显微镜（FE-SEM）在嵴水平分析线粒体超微结构的连续切片图像的3D重建新技术;通过这项技术，可以使用高效且可访问的方法获取高分辨率数据。使用超薄切片机制成的系列超薄切片可以半永久地存储在网格盒中，并在几年后多次重新成像41.FE-SEM因其能够提供高分辨率成像，高放大倍率和多功能性而作为各种研究领域的工具受到高度重视42。为了尝试以3D形式显示细胞器的精细结构，使用FE-SEM 43,44产生的背散射电子生成具有有用分辨率的串行2D图像堆栈的技术也可用于实现目标区域或其相关结构的高通量和多尺度成像，而无需特殊设备45.电荷伪影的产生直接影响采集图像的质量，因此保持较短的停留时间尤为重要。

因此，本研究详细阐述了该SEM技术中用于重建线粒体嵴46的3D结构的实验程序。具体来说，我们展示了使用Amira软件实现线粒体区域的半自动分割和数字化3D重建的过程，其中还包括使用传统的OTO标本制备方法44,47制作切片样品，使用超切片机切片完成切片收集，并通过FE-SEM获取顺序2D数据。

Protocol

1. 材料准备 在12mL的DMEM培养基（10%胎牛血清和100U / mL青霉素 – 链霉素）中培养2 x 106 Panc02细胞，并在5%二氧化碳和95%空气的气氛中保持在37°C和95%湿度48小时。 收集Panc02细胞，以28× g 离心2分钟，然后弃去上清液。确保样品具有适当的大小（1 x 107 个细胞），否则，以下固定和脱水步骤将无法正常工作。 加入1mL的2.5%戊二醛作为固定剂，将新?…

Representative Results

在细胞培养过程中（图1A），我们首先将胰腺癌细胞分为用完整培养基培养的对照组，（1S，3R）-RSL348（RSL3，铁死亡激活剂，100nM）组和RSL3（100nM）加铁抑素-149（Fer-1，铁死亡抑制剂，100nM）组。通过上述实验步骤，扫描电镜分别获取对照组、RSL3组和抑制剂组（RSL3+Fer-1组）的38张（补充图1）、43张（补充图2）和44?…

Discussion

这里介绍的方法是一个有用的分步指南，用于应用3D重建技术，该技术涉及将电子显微镜和图像处理技术应用于从连续超薄切片生成的2D断层扫描图像的堆叠和分割。该协议强调了可以通过细胞器超微结构的3D可视化来解决的2D图像的局限性，该图像具有高分辨率水平上结构的强可重复性和更高的精度的优点。更重要的是，这种3D可视化可以应用于肿瘤细胞，使病理机制的研究更加直接可靠。在这项…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

本研究得到了浙江省自然科学基金资助（Z23H290001，LY19H280001）;国家自然科学基金（82274364、81673607、81774011）;以及湖州市科技基金公益研究项目（2021GY49,2018GZ24）。感谢浙江中医药大学中医科学院医学研究中心公共平台的大力帮助、技术支持和实验支持。

Materials

(1S,3R)-RSL3 MCE HY-100218A
Acetone SIGMA 179124
Amira Visage Imaging
Aspartic acid MCE HY-42068
Dulbecco's modified Eagle’s medium Gibco 11995115
Ethanol Merck 100983
Ferrostatin-1 MCE HY-100579
Fetal bovine serum Gibco 10437010
Field emission scanning electron microscope HITACHI SU8010
Glutaraldehyde Alfa Aesar A10500.22
Lead nitrate SANTA CRUZ sc-211724
Osmium Tetroxide SANTA CRUZ sc-206008B
Panc02 European Collection of Authenticated Cell Cultures  98102213
Penicillin-streptomycin Biosharp BL505A
Phosphate Buffered Saline Biosharp BL302A
Pon 812 Epoxy resin SPI CHEM GS02660
Potassium ferrocyanide Macklin P816416
Thiocarbohydrazide Merck 223220
Ultramicrotome LEICA EMUC7
Uranyl Acetate RHAWN R032929 2020.2
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Tags

3D ReconstructionElectron MicroscopyUltrastructureMitochondriaPancreatic CancerOrganelle MorphologyOTO MethodSerial SectioningVisualization
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Qi, Y., Liu, Y., Huang, Y., Xiong, M., You, S., Wang, B., Gu, M. A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells. J. Vis. Exp. (196), e65290, doi:10.3791/65290 (2023).
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