The absolute calibrated ΔψM assay is based on fluorescence microscopy of the cationic dye (TMRM; tetramethylrhodamine methyl ester) together with an anionic, fluorescent plasma membrane potential indicator (PMPI; a bis-oxonol from the FLIPR plasma membrane potential kit; Molecular Devices). The technology back-calculates ΔψP and ΔψM that drive changes in the time courses of fluorescence intensities.

Fluorescence intensity changes of redistribution-type potentiometric probes reflect potentials distorted in time by their slow diffusion across the plasma membrane. The calibration algorithm models and cancels these effects. The algorithm also takes cell size, mitochondrial content, ΔψP, probe binding and auto- or background fluorescence into account to calculate ΔψM.

The electrostatic barrier model of ion
transport through the plasma membrane (see scheme on the right)
accurately describes the behavior of the probes in cells. This model
provides the calibration rate equation (below the cell). The ΔψP is
calculated by solving this equation for ΔψP. ΔψM is calculated
by the Nernst equation where [TMRM]_{M} and [TMRM]_{C}
are expressed by the rate equation and the total fluorescence. See definition of terms in the
calibration equations here (17).

In practice, the calculation of absolute millivolt values of ΔψM and ΔψP is performed by finding all required parameters in the calibration equations. An internal calibration protocol provides all of these information. The Membrane Potential Calibration Wizard of Image Analyst MKII calculates all required parameters and applies the calibration equations on the fluorescence traces.

*The internal calibration encodes the
absolute values of ΔψP and ΔψM in the fluorescence intensity time
courses. Importantly, this enables not only absolute millivolt value
readout, but also unbiased comparison of different samples. *

The calibration requires two axillary assays (measurement of mitochondria:cell volume fractions and the binding affinity/activity of TMRM to membranes). These assays rely on confocal microscopic recordings and image analysis in Image Analyst MKII. See protocols here. Alternatively, volume fractions may be known from literature. We found that the binding affinity of TMRM (expressed by aR') is in a narrow range between a variety of samples, therefore in many application..

*Image Analyst MKII is a one-of-a-kind solution for the measurement of the absolute magnitude of mitochondrial membrane potential (ΔψM) in intact cells.*Image Analyst MKII provides the complete and extended version of the plasma and mitochondrial membrane potential measurement technique developed by Akos Gerencser and colleagues (17). This is supported by protocols for data acquisition, image processing pipelines for measurement of fluorescence intensities in image data from a variety of formats and the intuitive Calibration Wizard dialog designed for easy and routine use for biologists with no biophysical or mathematical expertise required.

*Why to look ΔψM ?*

- ΔψM is a key bioenergetic parameter:
- the major component of the
proton motive force that determines the maximal available rate of ATP
formation in mitochondria
- knowledge of ΔψM helps to interpret
alterations in energy metabolism when matched with cell respirometry,
e.g. to identify uncoupling, or decreased activities of respiratory
complexes or ATP synthesis/transport
- high proton motive force is thought to be associated to increased production of free radicals

- the major component of the
proton motive force that determines the maximal available rate of ATP
formation in mitochondria
- Assaying ΔψM is relevant to research of:
- aging: efficiency of energy production, reactive oxygen species formation
- cancer: Warburg effect, cell-to-cell heterogeneity
- metabolism: uncoupling proteins, substrate oxidation pathways, substrate switching
- diabetes: ΔψM is a central component of the canonical pathway of insulin secretion

*Why most common fluorescence techniques fail to correctly measure
ΔψM ?*

**TMRM, TMRE non-quench mode (without using the Membrane Potential Calibration Wizard)**- The readout is a function of cell size, mitochondrial density, plasma membrane potential (ΔψP), probe binding and time
- Most often changes in ΔψP are
misinterpreted as changes in ΔψM
- relative to baseline measurement

**TMRM, TMRE, Rhodamine 123, DiOC6(3) quench mode**

- Relies on the assumption of a constant quench
limit
- The readout is a function of mitochondrial density
- relative
to baseline measurement
- A common mistake in flow cytometer
applications is that quench mode probes report mitochondrial mass and
not potentials
- Toxicity

- Relies on the assumption of a constant quench
limit
**JC1 emission ratio**

- The probe
accumulation is not an equilibrium process, therefore the readout is
dependent on ΔψP, time and surface to volume ratios.
- J-aggregates are
sensitive to oxidation
- The JC-1 emission ratio cannot be calibrated to millivolts because of the above confounding factors, and comparison of different samples can be easily misleading

- The probe
accumulation is not an equilibrium process, therefore the readout is
dependent on ΔψP, time and surface to volume ratios.

*What questions can be addressed with an absolute ΔψM assay?*

- Determination of ΔψM in millivolts in single or populations of cells
- Comparison of ΔψM in different samples (different cell types, pre-treatments, genotypes or individuals)
- Accurate comparison of relative changes between specimens with different amounts of mitochondria
- Measurement of changes of ΔψM when ΔψP is also changing
- Measurement of the heterogeneity of ΔψM
in cell populations
- E.g. in populations of type 2 diabetic and non-diabetic beta-cells (26)

- Kinetic analysis of oxidative phosphorylation
- ATP assay surrogate: ATP/ADP, phosphorylation potential are thermodynamically linked to ΔψM.