Fluorescence microscopy image analysis
automation - time series - physiology

TMRM PMPI technique - mitochondrial assay bis-oxonol and tetramethylrhodamine methyl ester (TMRM) Mitochondrial membrane potential assay
Absolute and Unbiased ΔψM Assay

   The assay is based on fluorescence microscopy of a cationic dye (tetramethylrhodamine methyl ester; TMRM) together with an anionic, fluorescent plasma membrane potential indicator (a bis-oxonol; aka PMPI). The technology back-calculates plasma membrane potential and mitochondrial membrane potential in millivolt values that drive changes in the time courses of fluorescence intensities. This enables not only absolute millivolt value readout, but also unbiased comparison of different samples.

The Theory
Theory of the TMRM/PMPI technique

How absolute millivolts calculated

See how effects of ΔψP, cell and mitochondrial geometry and fluorescence background accounted for to calculate mV values.

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In Practice
Membrane Potential Calibration Wizard

The Membrane Potential Calibration Wizard

This interactive dialog works hand in hand with the provided protocol to calibrate potentials without theoretical knowledge.

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See the technology in publications
  • In human stem cells (14)
  • In rat neurons (17)
  • In human β-cells (26)
Protocol
Image Analyst MKII protocols

Protocols

Protocols explaining experimental design, microscopy setup and image analysis

Protocols area