Methods to Evaluate Changes in Mitochondrial Structure and Function in Cancer
Abstract
:Simple Summary
Abstract
1. Introduction
2. Structural Techniques and Parameters to Characterize Mitochondria Health
2.1. Optical Microscopy
2.2. Electron Microscopy-Based Techniques
2.3. Evaluation of mtDNA Content or Integrity Using PCR
2.4. Extracellular Vesicle (EV) Secretion and Cargo
3. Functional Parameters to Understand Mitochondrial Health
3.1. Reactive Oxygen Species (ROS) Production
3.2. Mitochondrial Membrane Potential (ΔΨm)
3.3. Calcium Retention Capacity
3.4. Mitochondrial Bioenergetics: Glycolytic and Respiratory Capacity Measurements
Measures of ETC Complex Activity and the TCA Cycle
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Method | Applications | Strengths | Limitations | Ref |
---|---|---|---|---|
Widefield fluorescence and confocal microscopy | Mitochondrial morphology and dynamics, ΔΨm | Live-cell and time-lapse imaging Visualization of fluorescent proteins, dyes, and immunofluorescence staining | Resolution limited by diffraction (~1/2 λ) Fluorophore photobleaching | [25,78,79] |
Super-resolution microscopy (STED, FPALM, STORM, etc.) | Fine details of mitochondrial morphology and dynamics (e.g., cristae shape, width, etc.) | Superior resolution compared to the confocal Live-cell and time-lapse imaging | Requires specific fluorophores Often needs high laser powers | [33,34,35,39,80] |
Two-photon excitation fluorescence (TPEF) and fluorescence lifetime imaging | Mitochondrial dynamics, morphology and label-free redox state measurements, in vivo applications | Low background Superior light penetration depth Live-cell and time-lapse imaging Visualization of external probes or endogenous metabolites | Limited number of external fluorophores suitable for two-photon excitation | [44,45,81,82] |
Electron microscopy (EM) | Ultrastructural changes in mitochondrial shape, size, and components | Sub-nanometer resolution | Requires extensive fixation Limited ability to visualize markers Prone to artifacts | [58] |
Electron tomography | 3D information on mitochondrial ultrastructure | 3D structural information with high resolution | Requires extensive fixation “Missing wedge” because of the restricted tilt range Sample shrinkage due to high electron dose Limited ability to visualize markers | [65,83,84,85] |
Focused ion beam scanning electron microscopy (FIB-SEM) | 3D structural and compositional analysis | Label-free Resolution approaching EM | Not suitable for live cells Long image acquisition time (up to 60 h) | [71,72,73,74,75,76,77] |
Method | Strengths | Limitations | Refs |
---|---|---|---|
Quantitative PCR | High throughput High sensitivity and specificity Established methods Simply and widely used Real-time monitoring of target Versatile | Reliant on standard curves or normalization to reference gene Prone to PCR efficiency bias Sensitive to PCR inhibitors Sample quality requirements | [117,118,119] |
Digital PCR | High throughput Highly sensitive Absolute quantification Improved reproducibility Reduced PCR efficiency bias and PCR inhibition Highly accurate for low target concentrations No need for reference genes | Specialized equipment needed Cost prohibitive Extensive optimization required Sample quality requirements Narrow dynamic range Less accurate for high target concentrations | [117,118,127,128] |
Method | Strengths | Limitations | Refs |
---|---|---|---|
Seahorse Extracellular Flux Analyzer | High throughput Requires small quantities of cells Automated process/injections Compatible with broad range of cell and tissue types | Costly equipment setup Moderate optimization required Significant plate and reagents costs per experiments | [271,284,295] |
MitoXpress-Xtra® | Microplate-based assay High throughput Compatible with broad range of in vitro models Compatible with many plate readers | Moderate optimization required Significant plate and reagent costs per experiment Lower sensitivity Less well-established | [268,294] |
Orosboros-2k Oxygraph | Relatively cheap Low sample volumes required Reduced oxygen leakage from device compared to other electrodes Increased sensitivity compared to other electrodes | Measurements are not automated Labor-intensive Low throughput Time-intensive (hour-long sample reads) Lacks background controls | [277,296] |
Clark Electrode | Well-established method Reliable Mechanically robust Relatively low cost | Consumes oxygen Interference with various gases Labor-intensive maintenance | [294,297,298] |
EPR Oximetry | Higher sensitivity Relatively non-invasive Enables 3D oxygen mapping Minimal interference issues Spin probes are non-toxic & stable | Poor signal-to-noise ratio Motion artifacts Requires exogenous probe Longer acquisition times Limited penetration depth | [288,294] |
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Rickard, B.P.; Overchuk, M.; Chappell, V.A.; Kemal Ruhi, M.; Sinawang, P.D.; Nguyen Hoang, T.T.; Akin, D.; Demirci, U.; Franco, W.; Fenton, S.E.; et al. Methods to Evaluate Changes in Mitochondrial Structure and Function in Cancer. Cancers 2023, 15, 2564. https://doi.org/10.3390/cancers15092564
Rickard BP, Overchuk M, Chappell VA, Kemal Ruhi M, Sinawang PD, Nguyen Hoang TT, Akin D, Demirci U, Franco W, Fenton SE, et al. Methods to Evaluate Changes in Mitochondrial Structure and Function in Cancer. Cancers. 2023; 15(9):2564. https://doi.org/10.3390/cancers15092564
Chicago/Turabian StyleRickard, Brittany P., Marta Overchuk, Vesna A. Chappell, Mustafa Kemal Ruhi, Prima Dewi Sinawang, Tina Thuy Nguyen Hoang, Demir Akin, Utkan Demirci, Walfre Franco, Suzanne E. Fenton, and et al. 2023. "Methods to Evaluate Changes in Mitochondrial Structure and Function in Cancer" Cancers 15, no. 9: 2564. https://doi.org/10.3390/cancers15092564