Mitochondrial membrane potential assay - measurement of the apparent activity coefficient ratio - 1 - process

Parameters:

Name	#	Type	Description
Sensitivity factor	1	real	Factor for Otsu's threshold level. Lower values will take more objects as mitochondria
Mitochondrial ROI file Filename	2	string	*.roi file
Nucleus ROI file Filename	3	string	*.roi file

Description:

This pipeline is an accessory to the TMRM/FLIPR mitochondrial membrane potential measurement technique, and calculates a parameter required for calibration of mitochondrial membrane potential to millivolts. This parameter, the apparent activity coefficient ratio (aR’), expresses probe binding to mitochondrial membranes, differences in chemical activities between the cytosol and the mitochondrial matrix and the dilution of TMRM fluorescence by the intermembrane space (as given in J Physiol. 2012 590:12 2845-71).
Run this pipeline in a confocal microscopic time-lapse recording of TMRM fluorescence capturing the loss of fluorescence after complete mitochondrial depolarization.
After running the pipeline draw nuclear and mitochondrial ROIs in the respectively named image windows. Finally use the "Mitochondrial membrane potential assay - measurement of activity coefficient ratio - 2 - plot" pipeline to calculate the apparent activity coefficient ratio.
See protocol here: http://help.imageanalyst.net/protocols_ActivityCoefficientRatio.html
Version history:
V2: Sensitivity factor parameter has been added (pipeline function is identical to V1 when factor=0.5)
V3: Channel numbering was changed to match the V2 plotting pipeline
V4: ROI loading was added