Design Experiments with Comprehensive, Data-Driven Biological Insights

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Experiment Design Using UpDPC

The upgraded polarization-resolved differential phase contrast (UpDPC) microscopy technique is a powerful tool for tracking mitochondrial movement in response to cellular stressors. This method allows for high-resolution, label-free imaging of organelles, making it particularly suitable for studying mitochondrial dynamics under various conditions.

1. Objective

The primary objective of this experiment is to utilize UpDPC to monitor the movement of mitochondria in live cells subjected to different cellular stressors, such as oxidative stress, nutrient deprivation, and mechanical stress. This will provide insights into how mitochondria adapt metabolically to stress.

2. Materials and Methods

• Cell Lines: Use human iPS cells or HeLa cells for their well-characterized mitochondrial dynamics.
• Stress Induction: Apply oxidative stress using hydrogen peroxide (H₂O₂), nutrient deprivation by serum starvation, and mechanical stress through substrate stiffness variation.
• UpDPC Setup: Integrate UpDPC into a standard fluorescence microscope. Ensure the system is calibrated for high-speed imaging (over 100 Hz) and capable of achieving spatial resolution below 100 nm.
• Imaging Protocol: Capture mitochondrial movement before, during, and after stress application. Use time-lapse imaging to record mitochondrial dynamics.

3. Data Analysis

Analyze the acquired images using custom software to quantify mitochondrial movement parameters such as:

• Distance traveled
• Speed of movement
• Changes in mitochondrial morphology

Utilize kymographs to visualize mitochondrial trajectories and assess the impact of stress on their motility.

4. Expected Outcomes

This experiment is expected to reveal how different stressors affect mitochondrial movement and morphology, providing insights into metabolic adaptations. For instance, increased oxidative stress may lead to reduced mitochondrial motility, indicating a potential shift in metabolic pathways.

5. Significance

Understanding mitochondrial dynamics in response to stress is crucial for elucidating their role in cellular metabolism and health. This knowledge can inform therapeutic strategies for diseases associated with mitochondrial dysfunction.

References

• UpDPC microscopy study
• FOXP1 syndrome study