Fluorescence microscopy image analysis
automation - time series - physiology

The Membrane Potential Calibration Wizard

Mitochondrial Membrane Potential Assay

Step 1: The potentiometric paradigm

The assay can be performed on any epifluorescence (suitable for low-light level time-lapse imaging), confocal or two-photon system. A prototype experiment is exemplified below using human pancreatic beta-cells (published in (26)). The calibrant cocktails have been described in (17).

Step 2: Image Analysis

The mitochondrial membrane potential analysis can now be performed using an interactive protocol in the Primer Window by selecting Assays/Intensity and Ratio Measurements/ Mitochondrial membrane potential assay - worked example. Alternatively, select from a set of pre-configured pipelines in the main menu of Image Analyst MKII to process fluorescence time-lapse image recordings and automatically draw ROIs to extract fluorescence intensities. See tutorial image data for potentiometric image analysis here, and a video tutorial here.

Pre-configured pipelines in Image Analyst MKII

Human beta-cell with TMRM and PMPI

Left: Microscopic view-field of TMRM (red) and PMPI (green) fluorescence in pancreatic beta-cells.

Human beta-cell line scan

Right: Effect of channel alignment and image stabilization of the time lapse, demonstrated by a line scan

These image processing steps are included into the standard pre-processing pipelines for potentiometric calibrations.

Step 3: Calculation of millivolts using the Membrane Potential Calibration Wizard

The Membrane Potential Calibration Wizard guides the user through the calibration procedure. The user selects the experimental paradigm matching the recording, then points the ranges of calibrant additions on the fluorescence time courses, and finally enters required additional parameters. In the complete calibration paradigm only volumes of K+-based medium additions are required to perform the calibration.  

Eight ΔψP calibration paradigms are available supporting a variety of experimental designs. These differ in what calibration points are used and what assumptions are made. The complete calibration uses no assumption on any specimen-specific parameter.
  • Complete (temporally resolved K-steps)
  • Complete with known kP (K-steps)
  • Baseline & Zero
  • Baseline & K-equilibrium
  • K-equilibrium & Zero
  • K-steps with known [K+]i
  • Zero (fx=0)
  • Baseline & fP0
Three ΔψM calibration paradigms are available, that can be used together with any of the ΔψP calibration paradigms:
  • Complete
  • Complete (known k)
  • Baseline & MDC or CDC (known rest)
Membrane Potential Calibration Wizard

Step 4: Automation

Using pipeline automation the Membrane Potential Calibration Wizard can be executed completely automatically in each stage position or well of a microplate, analyzing thousands of individual cells in a single run. Millivolt calibrated data can be collected in Graphpad Prism from multiple conditions and experiments including pooling technical replicates.

Processed TMRM/PMPI image

As part of the automated pipeline processing, individual cells are identified by an automatic ROI drawing feature.

MMP pipeline

The menu-accessible pipelines are invisible for normal operation, and only key-parameters are shown in the main parameter bar. However, pipelines can be opened for editing and arbitrarily changed.

Theory Overview