Determination of Mitochondrial Morphology in Live Cells Using Confocal Microscopy

Summary

In this study, we describe a step-by-step protocol and emphasize the key details for determining morphological characteristics of mitochondria in live cells, including sample preparation, image acquisition, and data analysis. This method is commonly used to examine mitochondrial morphology for studying various conditions.

Abstract

The dynamic balance of mitochondrial fusion and fission directly contributes to mitochondrial homeostasis, which influences numerous cellular functions in addition to adenosine triphosphate (ATP) homeostasis. Therefore, assessing mitochondrial morphology under stress conditions is essential for mechanistic research. This study describes a detailed protocol for analyzing mitochondrial morphology, encompassing the preparation of a MitoTracker solution, staining of mitochondria, optimization of imaging parameters, and detection of morphological features. MitoTrackers are commonly used, cost-effective mitochondrion-specific dyes. However, some changes in mitochondrial morphology may occur owing to inappropriate handling, which can be unperceivable and fail to reflect the true state of mitochondria. Therefore, it is necessary to understand how to analyze changes in mitochondrial morphology using MitoTrackers. The protocol utilized SH-SY5Y cells stimulated with 1-methyl-4-phenylpyridinium iodide (MPP⁺) to illustrate the protocol of mitochondrial morphological analysis. Compared with control cells, MPP⁺-stimulated cells exhibited smaller and more fragmented mitochondria, with morphological parameters indicating decreased mitochondrial footprint. These results suggest that MitoTracker staining is an effective and feasible method for mitochondrial morphological analysis that (with minor modifications) can be applied to study various conditions.

Introduction

Mitochondrial morphology is maintained by the dynamic balance of mitochondrial fusion and fission, and hence, affects energy homeostasis and numerous other cellular functions, leading to various pathologies such as neurodegenerative diseases, cancer, and inflammation^1,2,3,4,5. To observe mitochondrial morphology, several mitochondrion-selective stains, including both probes and antibodies, have been developed for use with a confocal microscope. Some mitochondrion-specific probes, like tetramethylrosamine and rhodamine 123, function based on the mitochondrial membrane potential (MMP), which limits their applicability in studies of MMP-disrupting conditions^6,7. Conversely, antibody staining provides MMP-independent morphological assessment for examining mitochondrial morphology in fixed cells. Although a suitable stain allows for the assessment of mitochondrial morphology in fixed cells, examining mitochondrial morphology in live cells impaired by some inducers remains challenging. MitoTracker, a cell-permeable cationic and mitochondrion-specific fluorescent probe, offers a solution to this challenge, allowing staining of both live and fixed cells. This probe has been widely utilized to examine mitochondrial morphology in studies of various diseases^8,9,10,11. Nevertheless, some studies have reported issues such as high background signal and non-specific binding, even when the probe is utilized in strict accordance with the guidelines provided by the manufacturer. These observations highlight the importance of a thorough understanding of sample preparation and image acquisition for the successful analysis of mitochondrial morphology.

To facilitate analysis of mitochondrial morphology, various image processing programs and algorithms have been developed^{12,13,14,15,16}. In this study, we employed the macro tool Mitochondrial Network Analysis (MiNA) in ImageJ to analyze mitochondrial morphology¹³. This tool effectively identifies and characterizes morphological features of mitochondrial networks by calculating nine descriptive parameters, including the number of individuals (structures without any branches), number of networks (number of objects with at least one junction pixel), mean length (average length of all rods and branches), median length (median length of all rods and branches), length standard deviation (standard deviation of individual lengths), mean Network Size (the average number of branches per network), median Network Size (the middle value of the number of branches per network), network Size Standard Deviation (standard deviation of the number of branches per network), and the mitochondrial footprint (total signal area after background separation). In this protocol, the mitochondrial footprint was selected as a representative parameter.

The objective of this study is to provide a step-by-step protocol for mitochondrial morphological analysis, focusing on key details. We used MPP⁺-induced SH-SY5Y cells to demonstrate this protocol. MPP⁺ inhibits complex I of the mitochondrial respiratory chain in dopaminergic neurons, resulting in damage to mitochondrial morphology and function^8,17. The morphological analysis protocol described in this study enables the acquisition of high-resolution images and the assessment of relevant parameters to define morphological features of mitochondrial networks.

Protocol

1. Preparation

1. Dissolve MPP⁺ with dimethyl sulfoxide (DMSO) to make a 1 mM stock solution and store it at -30 °C in the dark (see Table of Materials).
2. Dissolve 50 µg of MitoTracker (MitoTracker Red CMXRos) in 94 µL of DMSO to make a 1 mM stock solution. Aliquot the stock solution into several microcentrifuge tubes and store protected from light at -30 °C until further use.
3. Incubate SH-SY5Y cells in flasks with Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) in a humidified environment with 5% CO₂ and 95% air at 37 °C. For passaging, detach the cells with 0.05% trypsin solution for 1 min and centrifuge at 100 × g for 3 min.

2. MPP⁺ stimulation

1. Seed SH-SY5Y cells on confocal dishes with a glass bottom and maintain them in DMEM/F12 medium supplemented with 1% (v/v) FBS overnight.
2. Replace the culture medium, then incubate the cells with or without MPP⁺ for 24 h.

3. MitoTracker staining

1. Prewarm DMEM/F12, then dilute the MitoTracker stock solution with DMEM/F12 to obtain a 50 nM working solution. Keep the solution protected from light during the whole procedure.
2. Remove the cell culture medium and wash the confocal dishes 2x with fresh DMEM/F12.
3. Incubate cells in each dish with 1 mL of the MitoTracker working solution for 15 min at 37 °C in the dark.
4. Remove the MitoTracker working solution and wash 2x with DMEM/F12.
5. Add 1 mL of DMEM/F12 to each dish and incubate in the cell incubator before imaging.

4. Imaging parameters for confocal microscope

1. Preequilibrate the microscope for subsequent operations. Under the acquisition panel, adjust the light path-related parameters by setting the excitation and emission wavelengths.
2. Set the parameters as follows: pinhole size, 1.2 AU; pixel scan size, 1,024 x 1,024; pixel dwell time, 2.4 µs (average 2x) (keep it consistent for calibration purposes).

5. Optimizing the imaging parameters

CAUTION: Avoid eye exposure to both direct and scattered radiation from a visible and/or invisible laser.

1. Use the halogen light to focus on the cells using a plan apochromatic 60x objective lens (1.4 NA) to avoid phototoxicity.
2. Adjust the laser power and high voltage (HV) to just below the saturation level, set the zoom factor to 3 and then capture the images.
3. Move the region of interest and focus on the cells to capture images without changing any settings.

6. Data analysis

NOTE: Data were analyzed using ImageJ preinstalled with the MiNA plug-in¹³. To obtain accurate relevant measurements, images should capture independently resolved mitochondria with clean and bright staining.

1. To enhance image quality prior to binary transformation and skeletonization, open the image and conduct preliminary processing. Use the ROI tool to select the area of a single cell. Apply the Process | Filters | Unsharp Mask function to enhance sharpness, and use the Process | Enhance Contrast feature to reduce noise over-amplification. To remove salt and pepper noise specifically, use the Process | Filters | Median function (see Supplemental Figure S1).
2. To construct a simplified morphological model to calculate descriptive parameters, convert the image to binary via thresholding (click on Process | Binary | Make Binary), and then skeletonize the binary image using the Skeletonize function (click on Process | Binary | Skeletonize) (see Supplemental Figure S2).
3. Group all pixels within the skeleton using the Analyze | Skeleton | Analyze Skeleton (2D/3D) function. Simplify the analysis by using the MiNA tool to calculate nine descriptive parameters in the skeletonized image (see Supplemental Figure S3).

Results

This protocol outlines a detailed, step-by-step procedure for mitochondrial morphological analysis through three interdependent optimization modules: standardization of specimen preparation protocols, systematic optimization of optical parameters for confocal microscopy imaging, and computational image processing guidelines. Optimal sample preparation protocol is critical for obtaining reliable mitochondrial imaging data. Systematic evaluation of MitoTracker Red CMXRos concentrations demonstrated concentration-dependent ...

Discussion

The present study establishes a systematic methodology for analyzing mitochondrial morphology in live cells under stress conditions. Crucial procedural steps in mitochondrial imaging involve: an appropriate concentration of MitoTracker probes, thoroughly washing confocal dishes prior to image acquisition, minimizing disturbances to the cellular state, and setting suitable imaging parameters.

Using higher concentrations of MitoTracker can induce cytotoxicity, rendering mitochondria sensitive to...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-methyl-4-phenylpyridinium iodide (MPP+)	Macklin	M875357	Used to induce oxidative stress
Confocal dish	NEST	801001	Used to culture the cells
Confocal microscope	Nikon	C2	Used to capture images
Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12	ThermoFisher Scientific	11320033	Used to provide nutrition to cells
Dimethyl sulfoxide (DMSO)	Beyotime	ST038	Used to dissolve the mitotracker probe
Fetal bovine serum (FBS)	ThermoFisher Scientific	A3161001C	Used to provide nutrition to cells
ImageJ	National Institutes of Health	ImageJ	Used to analyze mitochondrial morphology
MitoTracker Red CMXRos	ThermoFisher Scientific	M7512	Used to visualize mitochondria
Phosphate Buffer Saline (PBS)	ThermoFisher Scientific	C10010500BT	Used to wash the cells on confocal dishes
SH-SY5Y cell	ATCC	CRL-2266	Used to demonstrate the workflow of analyzing mitochondrial morphology
TI-SH-U Stage Adapter	Nikon	MEC59110	Used to secure specimens