Determination of Mitochondrial Morphology in Live Cells Using Confocal Microscopy

Manna Lin; Jiakang Wang; Zihong Xian; Chunyuan Zeng; Jiangping Xu

doi:10.3791/68167

JoVE Journal
Neuroscience

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Neuroscience

Determination of Mitochondrial Morphology in Live Cells Using Confocal Microscopy

使用共聚焦显微镜测定活细胞中的线粒体形态

Full Text

1,473 Views

06:57 min

July 3, 2025

DOI: 10.3791/68167-v

Manna Lin^1,2, Jiakang Wang¹, Zihong Xian¹, Chunyuan Zeng¹, Jiangping Xu¹

¹NMPA Key Laboratory for Research and Evaluation of Drug Metabolism & Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences,Southern Medical University, ²Central Laboratory,Southern Medical University

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents a detailed protocol for assessing mitochondrial morphology in live SH-SY5Y cells, relevant for pharmacological studies, especially in Parkinson's disease. The method emphasizes accessibility and lower costs through the use of fluorescence imaging with MitoTracker staining, avoiding complex techniques like electron microscopy.

Key Study Components

Area of Science

Neuropharmacology
Mitochondrial function
Cell imaging techniques

Background

Challenges in lab cell analysis persist despite manufacturer guidance.
This study addresses these by outlining a step-by-step protocol for mitochondrial morphology assessment.
The focus is on reducing costs and enhancing accessibility for researchers.

Purpose of Study

To develop a reproducible method for analyzing mitochondrial morphology in live cells.
To facilitate understanding of drug compound relationships with mitochondrial function in neurodegenerative conditions.
To compare the effectiveness of simple staining methods against more complex imaging techniques.

Methods Used

The primary platform used is live cell culture.
SH-SY5Y neuroblastoma cells are employed as the biological model for examining mitochondrial response to pharmacological agents.
Image acquisition involves fluorescence microscopy and open-source software for data analysis.
Key steps include cell detachment, MitoTracker staining, and image processing techniques such as skeletonization.

Main Results

The developed protocol allows for improved visualization and analysis of mitochondrial morphology.
MPP+ treatment was shown to significantly disrupt mitochondrial structure in treated cells compared to controls.
Fluorescence intensity and background noise are key factors in imaging quality.

Conclusions

This study establishes a reliable and cost-effective approach for imaging mitochondrial morphology in live neurons.
The method enhances understanding of mitochondrial dynamics in drug response, particularly in neurodegenerative disease models.

Frequently Asked Questions

What are the advantages of using the MitoTracker staining method?

MitoTracker staining is a straightforward and cost-effective approach for assessing mitochondrial morphology, avoiding the complexities of electron microscopy.

How are SH-SY5Y cells prepared for imaging?

The SH-SY5Y cells are cultured, detached using trypsin, and then stained with MitoTracker before imaging with a confocal microscope.

What types of data are obtained from this imaging method?

This method provides data on mitochondrial morphology and fluorescence intensity, which can inform on cell health and response to treatments.

How long does the entire staining and imaging process take?

After cell preparation, the staining and equilibration steps take approximately 30 minutes before imaging can commence.

Can this method be adapted for other cell types?

Yes, the protocol can be optimized for other cell types by adjusting staining conditions and imaging parameters accordingly.

What limitations should be considered when using this method?

Potential limitations include background fluorescence interference and the need for careful calibration to avoid photobleaching during imaging.

在这项研究中，我们描述了一个循序渐进的方案，并强调了确定活细胞中线粒体形态学特征的关键细节，包括样品制备、图像采集和数据分析。这种方法通常用于检查线粒体形态以研究各种情况。

我们的研究重点是基础神经药理学。具体来说，我们研究药物分子的工作原理。我们旨在了解帕金森病中潜在药物化合物与线粒体功能之间的关系。

先前的研究回顾了长期存在的实验室细胞分析挑战，即使有制造商指南也是如此。为了解决这个问题，我们概述了评估实验室细胞中线粒体形态的详细分步方案。我们的协议使用简单的线粒体染色、荧光成像和开源软件。

View the full transcript and gain access to thousands of scientific videos

Explore More Videos

JoVE 本月刊第 221 期线粒体活细胞共聚焦显微镜 ImageJ MitoTracker 线粒体形态学