Mitochondrial membrane potential assay (TMRM/FLIPR) with masking dead cells - for groups of cells

Parameters:

Name	#	Type	Description
Channel Number, TMRM	1	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number.
Channel Number, FLIPR	2	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number.
Dead mask: before FLIPR calibration frame	3	integer	Last frame of baseline or before plasma membrane potential is depolarized during calibration.
Dead mask: percentile of FLIPR fluorescence at zero potential	4	real	Percentile of fluorescence intensity of calibration frames. Lower value will apply a lower threshold to consider a cell depolarized on baseline. Set it to 100 to disable.
Background level (percentile)	5	real	Background is calculated as frame-by-frame median of those pixels that are the darker than this percentile of image histogram for the duration of the entire recording.
Spectral Unmix Coefficient Matrix	6	string	To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix.
Image stabilizer compares to first frame	7	boolean	“Yes” is the fastest way of image stabilization. Turn it off if the image changes a lot during the time lapse, and it is not possible to compare later frames to the first frame any more.
Image stabilizer period (frames)	8	integer	1 if comparing to the first frame. Use more frames if not comparing to the first frame and the view field drifts slowly.
ROIs: Cell diameter (pixels)	9	integer	Diameter of the cell in pixels in the combined TMRM+FLIPR projection image. Cells ranging around this size will be selected.
ROIs: Margin around cells (pixels)	10	integer	Cell boundaries will be extended by this amount.
ROIs: Debris cutoff (percentile)	11	real	This percentile of the image histogram sets the intensity value where the maximum of the Look Up Table (LUT) is scaled. Use -1 to override this with fixed value set below at "Max value".
ROIs: Minimum cell fluorescence (%)	12	real	Cells dimmer than this in filtered rescaled TMRM+FLIPR projection images will be rejected. Increase this value if debris dimmer than the cells is detected.
ROIs: Sensitivity of cell boundaries (% of max fluorescence)	13	real	For "Bound locally", the boundary of each object is determined at this % of the maximal intensity of the object relative to its neighborhood. For "Bound uniformly" this is a pixel intensity value.
Membrane potential calibration action	14	string	Select an action to be performed by the Membrane Potential Calibration Wizard
Calibration configuration file name (*.ips)	15	string	Configuration file previously saved from the Membrane Potential Calibration Wizard
Output Excel Data save file name (*.xlsx)	16	string	Excel Data window output will be created and saved using this name.

Description:

This pipeline performs all image processing tasks required for calibration of a TMRM / FLIPR (tetramethylrhodamine methyl ester and FLIPR plasma membrane potential assay kit; aka PMPI, plasma membrane potential indicator) fluorescence time lapse recording to millivolts. Processed images and calculated ROIs are passed to the Membrane Potential Calibration Wizard. Using pre-defined configuration, this pipeline is capable of unsupervised processing of multi-stage position mitochondrial membrane potential assays.

The pipeline corrects for misalignment between channels on the frame-by-frame basis, subtracts background, performs spectral unmixing with pre-defined coefficients, and stabilizes the time lapse (registers frames in time). To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used.
To adjust masking dead cells find the frame number of the last frame before the plasma membrane potential calibration was started (K-steps or complete depolarization). If the experiment included strong depolarization of plasma membrane potential before calibration, determine the frame number before that happened. Set this value at “Dead mask: before FLIPR calibration frame”. To tune how strongly are depolarized/dead cells removed provide a percent value at “Dead mask: percentile of FLIPR fluorescence at zero potential”. Lower values will remove more cells.
To calculate the spectral crossbleed matrix use the "Mitochondrial membrane potential assay - measurement of spectral crossbleed" pipeline, or the related Primer Window/Assays protocol.

The ROIs are generated automatically using blurred temporal maximal intensity projection image of the summed FLIPR+TMRM image to detect colonies or groups of cells. This approach is also useful if cells in a confluent culture move a lot during the recording. The calibration is not affected by the amount of background recorded in the ROI.
Calibration can be performed by manual configuration of the wizard, or using a previously saved configuration file to provide automation.
The Membrane Potential Calibration Wizard implements the calibration technique published by Gerencser et al. in J Physiol. 2012 590:12 2845-71. See more practical details in PLoS One. 2016 Jul 12;11(7):e0159199.

Version history:
V2
Segment welding has been enabled and separation of segments has been disabled.
No clipping during intensity scaling for seed calculation.
Intensity classifiers are applied to the seeds, as well as to the secondary segmentation.
Improved background subtraction
V3
Updated description
V4
Fixed pipeline parameter for ROIs: Sensitivity of cell boundaries (% of max fluorescence)
Adjusted ROI drawing to follow smoothed boundaries. This fixes a problem of finding too few ROIs.