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ΔF/F0

TMRM Complete Calibration ( IATMRMComplete )

Parameters:

Name	Short Name	Type	Description
Range definitions in time courses	rangedef	string	Defines whether the ranges given below refer to frames or seconds in the recordings
Baseline Range	BaselineRange	string	Defines the range of the baseline (in the above defined time units). Longer baseline results more accurate calibration.
VF (mitochondria:cell volume fraction)	VF	real	Confocal stereologic volume fraction of mitochondria (inner boundary membrane) to whole cell (including nucleus)
VFM (matrix:mitochondrion volume fraction)	VFM	real	EM stereologic volume fraction of matrix to mitochondrion (inner boundary membrane)
aR` (matrix:cytosol apparent activity coefficient ratio)	aR	real	Apparent activity coefficient ratio measured by confocal microscopy
MDC Range	MDCRange	string	The range of Mitochondrial Depolarization Cocktail addition (in the above defined time units). This range will be narrowed by differentiation kernel size and delay time.
MDC Delay (s)	MDCdelay	real	Delay time after starting Mitochondrial Depolarization Cocktail treatment to allow mitochondrial membrane potential to discharge completely (in seconds).
CDC Range	CDCRange	string	The range of Complete Depolarization Cocktail addition (in the above defined time units).
Take the minimum of CDC Range	CDCMin	boolean	If set yes, the minimum FLIPR intensity within the CDC Range is taken as zero calibration value (its error is the local SD of the range). Otherwise mean is calculated.
fT0-fTX Allowed Range	fT0MinusfTXRange	string	Trace selection criterion: the minimum and maximum allowed value of fT0-fTX
Quality control based on MDC calibrates to zero	QCMDC	boolean	Removes traces where MDC does not calibrate to zero, or noisy. Set threshold below.
Limit smallest offset from zero during MDC (mV)	QCMDCdeviation	real	Traces where the absolute mean of the last third MDC Range is larger than this number are removed. Zero value disables checking. Applied when "Quality control based on MDC calibrates to zero" is enabled.
Limit standard deviation during MDC (mV)	QCMDCSD	real	Traces where the SD of the entire MDC Range is larger than this number are removed. Zero value disables checking. Applied when "Quality control based on MDC calibrates to zero" is enabled.
Quality control by propagated error of baseline	QCMMPrest	boolean	Removes traces where the estimated SE of baseline is greater than the threshold below. Requires error propagation.
Limit SE of baseline (mV)	QCMMPrestMaxSE	real	Traces where the SE of resting ΔψM parameter is larger than this number are removed.
Limit baseline instability (SD mV)	QCMMPbaseStabilitySD	real	Traces where the SD calculated for the entire calibrated ΔψM baseline in time is larger than this number are removed.
kT Allowed Range	kTRange	string	Trace selection criterion: the minimum and maximum allowed value of kT
Maximum allowed DTE	DTEMax	real	Threshold value to eliminate the early steep part of TMRM decay, where ΔψM may not be fully depolarized. This is a criterion for the exact length of data to be used for regression.
Range of regression ends at zero DTE	TruncateRegressionAtZero	boolean	Criterion for the exact length of data to be used for regression.
TMRM r^2 Min	TMRMr2Min	real	Trace selection criterion: the minimum allowed value of r^2 for the TMRM-regression.
Filter regression points to the straightest section	FilterRegression	boolean	In addition to the above constrains, the most linear middle section of the DTE-FTE diagram is used for regression. Turn off "Range of regression ends at zero DTE" if using this method, and use zero for "MDC Delay (s)".
Tolerance for straightness	StraightnessTolerance	real	Maximum allowed fold change in derivative where the linear fit is performed.
Use K-steps instead of CDC to calculate ft0	forcezero	boolean	Uses TMRM fluorescence and calculated ΔψP corresponding to K-steps to calculate ft0 instead of measuring it in the CDC range. Use this option of measurment of ft0 is problematic for some reason.

Description:

This method calculates mitochondrial membrane potential (ΔψM) time course by determining all calibration parameters from a single time lapse.
Required experimental protocol: any time lapse, followed by mitochondrial depolarization resulting a well resolved decay curve of TMRM fluorescence and finally complete depolarization.
Determination of resting mitochondrial membrane potential requires baseline recording. Longer baseline results more accurate determination. If the baseline is not steady, use the "Do not assume equilibrium baseline" switch in the Constants tab.
Mitochondria need to be substantially polarized before addition of MDC. Cells have to be able to maintain some ΔψP during MDC addition. Very depolarized ΔψP leads to inaccurate or impossible calibration.