Live-imaging of Mitochondrial System in Cultured Astrocytes
November 16th, 2021
This article describes the method for mitochondrial time-lapse imaging of astrocyte cultures equipped with MitoTimer biosensor and the resulting multiparametric analysis of mitochondrial dynamics, mobility, morphology, biogenesis, redox state, and turnover.
This protocol greatly maximizes the power of microscopy as a method to track mitochondrial dynamics within individual cells over long acquisition periods. Unlike time-lapse on a fixed group of cells, or on one cell at a time, normalization to a baseline for each measured criterion in each cell controls for the heterogeneity between cells. With an microscope equipped with automatized platform and viral vector adapted, we can use this protocol for every type of cells.
Begin by plating 20, 000 to 25, 000 cells per square centimeters in multi-well dishes and store them at 37 degrees celsius under an atmosphere containing 5%carbon dioxide for the rest of the experiment. At eight days in vitro, add 0.6 pico grams of P24 antigen per cell of lentiviral vector coding for mitochondrial biosensor MitoTimer diluted in phosphate buffered saline. At 11 days in vitro, perform two washes with pre-warmed sterile PBS and add fresh astrocyte medium without phenol red.
Assess the Astrocytic mitochondrial system at least three to five days after the lentiviral infections with LV-G1 MitoTimer. Select five astrocytes per well with a mitochondrial network expressing sufficient levels of LV-G1 MitoTimer using a magnification of 40 times. Taking care to select astrocytes flat and large as possible and not located in clusters of cells.
Then capture fluorescence images using sequential excitation at 490 nanometers for the green channel and 550 nanometers for the red channel with green and red fluorescent signals and using a magnification of 150 times for each coordinate. Select the first frame for red and green channels for each image sequence by clicking on ND Processing and select frame. Then merge the red and the green channels by selecting conversions and merge channel.
To correct image shading, select pre-processing, and then auto shading correction. Apply the rolling ball algorithm by selecting pre-processing and rolling ball. To generate binary masks for each mitochondrion, select segmentation and threshold.
Remove any objects truncated by the border by selecting binary processing and touching border. Select measurement, and then object area, EEQ diameter, length, width, roughness, circularity, or elongation to measure surface area, diameter, length, width, roughness, circularity, or elongation respectively. Compose a group with these measurements and rename it as MOFO data.
Then select measurement, and then select mean intensity to measure the mean green and red intensities. And ratio to measure the red by green ratio. Now composer group with these measurements and rename it as ratio data.
Finally export the table to a CSV file by selecting reference and table to CSV, and then save the GA 3 script of analysis by selecting Save As.Begin by opening the tracking module in NIS and selecting view, analysis and tracking. Click on define new ROI. With the automatic detection tool, select 25 to 50 mitochondria on the first image of the image sequence, and then click on track auto detected ROIs analyze.
If necessary, delete the incorrect ROI tracks and export the table to a CSV file. Manually log transform the measurements before processing. For each analysis and timeframe manually normalize the resulting data by the mean obtained in the reference acquisition.
Then perform statistical analysis using a two way matched a Nova. Primary culture of astrocytes infected with LV-G1 MitoTimer exhibited reduced balanced and oxidized mitochondrial networks with different levels of fragmentation. Before hydrogen peroxide treatment, astrocytes expressing LV-G1 MitoTimer showed heterogeneous mitochondrial size in various green and red fluorescence intensities.
The mitochondrial morphology of astrocyte cultures was fragmented after six hours of incubation with hydrogen peroxide. It was even more apparent 12 hours after the treatment without their diameters with since sphericity reduction. Concerning redox state and turnover, three hours after a hydrogen peroxide treatment, the proportion of green mitochondria increased in astrocytes.
Concerning the dynamics and mobility three hours after treatment. All the criteria were transiently increased. This technique is suitable for many different questions.
For example, in the context of neurodegenerative disease, we're investigating the estrocytic toxicity of different molecules secreted in the brain.
Overview
This article details a method for mitochondrial time-lapse imaging in astrocyte cultures utilizing the MitoTimer biosensor. It explores mitochondrial dynamics, including mobility, morphology, biogenesis, redox state, and turnover due to various treatments.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Imaging Techniques
Background
- Astrocytes play a crucial role in brain function and health.
- Mitochondrial dynamics are essential for cellular metabolism and signaling.
- Investigating these dynamics provides insights into neurodegenerative diseases.
- Tracking individual cells over time offers greater detail in analysis.
Purpose of Study
- To establish a reliable protocol for analyzing mitochondrial behavior in astrocytes.
- To compare mitochondrial properties before and after hydrogen peroxide treatment.
- To highlight the technique's applicability for various research questions.
Methods Used
- This study employs a cell culture platform for astrocyte imaging.
- Astrocytes are infected with a lentiviral vector coding for MitoTimer.
- The fluorescence imaging protocol includes capturing signals at specific wavelengths.
- Key timelines include 8 days in vitro for transduction and assessments at various intervals post-treatment.
Main Results
- A reduction in mitochondrial integrity was observed following oxidative stress.
- Heterogeneity in mitochondrial size and redox state was noted before and after hydrogen peroxide treatment.
- Dynamic behaviors such as mobility and morphology exhibited varying responses to treatment.
Conclusions
- This study effectively demonstrates a method to quantify mitochondrial dynamics in astrocytes.
- The findings offer insights into the role of oxidative stress on astrocytic health.
- Such methodologies could be pivotal in understanding neurodegenerative mechanisms.
Frequently Asked Questions
Executive Industry Relevance
This method enables longitudinal tracking of mitochondrial dynamics in astrocytes, supporting target validation in neurodegenerative disease models by linking oxidative stress to measurable changes in mitochondrial morphology and redox state. It provides quantitative, single-cell resolution data that enhances predictive confidence in early discovery by de-risking mechanistic hypotheses about glial contributions to pathology. The approach is applicable to screening mitochondrial modulators in disease-relevant systems, offering translational continuity from in vitro findings to preclinical validation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by quantifying mitochondrial fragmentation and redox shifts in astrocytes under oxidative stress.
- Operational Value: Enables functional target validation through longitudinal imaging of mitochondrial mobility and biogenesis in live cells.
- Predictive Value: Supports portfolio triage by identifying compounds that normalize mitochondrial dynamics in disease-relevant glial models.
Screening & Assay Development
- Assay Readiness: Generates standardized mitochondrial morphology and intensity metrics for high-content screening of neuroprotective agents.
- Quantitative Outputs: Provides red-to-green ratio and morphological parameters (area, length, circularity) as reproducible endpoints for compound screening.
- Scalability: Compatible with automated microscopy platforms for multi-well imaging and batch analysis of mitochondrial responses.
Translational & Preclinical Research
- Disease Relevance: Models astrocytic mitochondrial dysfunction implicated in neurodegeneration, enabling mechanistic de-risking of targets.
- Translational Continuity: Bridges discovery-phase imaging with preclinical validation by tracking mitochondrial turnover and redox state over time.
- Risk-Adjusted Decisions: Informs go/no-go criteria based on normalization of mitochondrial networks following treatment.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target hypothesis testing to lead identification, providing mechanistic readouts that inform compound selection and optimization in neurodegenerative disease programs.
- Discovery Biology: Supports hypothesis testing by linking mitochondrial morphology changes to oxidative stress in astrocytes.
- Screening: Enables assay readiness through standardized mitochondrial biosensor expression and imaging parameters.
- Analytics: Delivers quantitative measurements (mean intensity, ratio, object morphology) for comparing experimental conditions across timepoints.
- Translational Research: Connects to preclinical work by modeling redox state and turnover changes relevant to neurodegeneration.
- Enterprise Reuse: Establishes a reusable imaging platform for studying mitochondrial dynamics across multiple cell types and disease models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing mechanistic ambiguity in glial mitochondrial responses to stress.
- Operational Value: Ensures reproducibility via baseline normalization and automated image processing workflows.
- Strategic Value: Improves capital efficiency by enabling early de-risking of targets through phenotypic screening in astrocytes.
- Portfolio Impact: Supports risk-adjusted advancement by identifying compounds that rescue mitochondrial network integrity.
Implementation Considerations
- Requires expertise in live-cell imaging, lentiviral transduction, and mitochondrial biosensor validation.
- Dependent on automated microscopy platforms with sequential excitation and environmental control.
- Necessitates cross-team standardization of image acquisition and analysis protocols for reproducible results.
- Involves adaptation considerations for different astrocyte sources or disease models.
- Limited by the need for prolonged culture periods and precise timing post-infection for optimal biosensor expression.
Biopharma FAQs
Chapters in this video
0:04
Introduction
0:44
Rat Hippocampal Astrocyte Primary Culture
1:25
Long-Term Monitoring of the Mitochondrial System
2:13
Analysis of Individual Mitochondrial Morphology and LV-G1-MitoTimer Ratio
3:54
Analysis of Mitochondrial Motility
4:28
Data Transformation, Normalization, and Statistical Analysis
4:51
Results: Analysis of the Effects of H2O2 on the Mitochondrial System of Astrocytes
5:53
Conclusion
