Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Emery L. Ng; Ashley L. Reed; Christopher B. O&#8217;Connell; Nathan N. Alder

doi:10.3791/65561

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

使用活细胞STED成像可视化神经元细胞模型中的线粒体内膜超微结构

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08:48 min

June 30, 2023

DOI: 10.3791/65561-v

Emery L. Ng¹, Ashley L. Reed², Christopher B. O’Connell¹, Nathan N. Alder²

¹Center for Open Research Resources and Equipment,University of Connecticut, ²Department of Molecular and Cell Biology,University of Connecticut

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Overview

This study presents a protocol for the propagation, differentiation, and staining of SH-SY5Y cells and primary rat hippocampal neurons. The aim is to visualize and analyze mitochondrial ultrastructure using stimulated emission depletion (STED) microscopy, providing insights into the structural dynamics of mitochondria in living cells.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Mitochondrial Research

Background

Mitochondria's structure and function are crucial for cellular health.
Traditional microscopy is limited in resolution for ultrastructural analysis.
Super resolution microscopy allows for more detailed imaging of mitochondrial features.
Understanding mitochondrial changes can provide insights into aging and dysfunction.

Purpose of Study

To establish a reliable method for studying mitochondrial ultrastructure in live cells.
To enable researchers to analyze dynamic changes in mitochondrial morphology.
To provide a foundation for investigating mitochondrial responses to various treatments.

Methods Used

Cell culture and differentiation of SH-SY5Y neuroblastoma cells.
Preparation of primary rat hippocampal neurons.
Utilization of STED microscopy for high-resolution imaging.
Key steps include cell thawing, coating cover slips, and staining with PKMO.
Image acquisition and analysis through specific software and plugins.

Main Results

Successful visualization of mitochondrial structures at nanoscale resolution.
Insights into cristae architecture and protein distribution within mitochondria.
Quantitative analysis of changes in mitochondrial features under physiological conditions.
Potentially significant findings regarding the response of mitochondria to pharmacological compounds.

Conclusions

The study demonstrates a valuable technique for investigating mitochondrial dynamics in living cells.
Contributes to understanding the role of mitochondria in cellular aging and health.
Implications for therapeutic interventions aimed at restoring mitochondrial function.

Frequently Asked Questions

What are the advantages of using SH-SY5Y cells in this study?

SH-SY5Y cells are a well-established model for neuronal studies, providing a suitable environment for differentiating and examining mitochondrial dynamics.

How are the primary rat hippocampal neurons prepared for imaging?

They are cultured and treated with specific media conditions to promote healthy differentiation and later imaged using the STED microscopy technique.

What types of data can STED microscopy provide?

STED microscopy allows for the visualization of mitochondrial ultrastructure and detailed analysis of protein localization within the inner mitochondrial membrane.

Can the methodology be adapted for other cell types?

Yes, the protocol can be adapted to other neuronal or non-neuronal cell types interested in studying mitochondrial morphology.

What limitations are associated with this imaging technique?

STED microscopy requires specific preparation and can be technically challenging, necessitating precise calibration of equipment for optimal results.

该协议提供了培养的SH-SY5Y细胞和原代大鼠海马神经元的繁殖，分化和染色的工作流程，用于使用受激发射耗竭（STED）显微镜进行线粒体超微结构可视化和分析。

我们的研究计划侧重于线粒体的结构和功能方面，特别是与衰老相关的功能障碍。为此，我们使用广泛的生物物理、生化和结构技术来探索线粒体衰老的基本机制，并开发治疗干预措施来保持和恢复线粒体健康。线粒体的功能和结构是密不可分的。

标准光学显微镜技术适用于测量大规模线粒体特征，如基本形态和网络结构。分析线粒体超结构或特征需要比传统光学显微镜更高分辨率，通常使用电子显微镜或固定样品断层扫描来完成。超分辨率显微镜是一种基于荧光的成像技术，可测量超出衍射极限的特征。

对于线粒体，这包括纳米级蛋白质分布和嵴结构的测量。这里描述的稳定成像方案允许研究人员测量活细胞中内膜的详细结构。活细胞成像使我们能够在生理相关条件下观察和定量分析嵴的复杂特征，而无需固定样品。

这将使研究人员对超结构特征中发生的动态变化及其对压力源和药理化合物的时间响应提供新的见解。