Mitochondrial membrane potential assay (TMRM/FLIPR) with post-hoc absorbance classifier

Parameters:

Name	#	Type	Description
Channel Number, TMRM in MD Open #1	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 in MD Open #1	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.
Channel Number, bright field image for absorbance classifier in MD Open #2	3	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number. This channel needs a blank and drark current reference images.
Channel Number, nuclear stain fluorescence in MD Open #2	4	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number.
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: Nucleus diameter (pixels)	9	integer	Diameter of the nucleus in pixels. Nuclei ranging around this size will be selected.
ROIs: Cell diameter (pixels)	10	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)	11	integer	Cell boundaries will be extended by this amount.
ROIs: Debris cutoff (percentile)	12	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 (%)	13	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.
Absorbance scaling Min value	14	real	Pixel intensity for the minimum of the Look Up Table. This affects visualization only.
Absorbance scaling Max value	15	real	Pixel intensity for the maximum of the Look Up Table. This affects visualization only.
Absorbance percentile of ROI means to keep an ROI	16	real	Cells with lower absorbance than this threshold given in percentiles of ROI means will be rejected.
FLIPR response classifier Frame Range:	17	string	Use this frame range to gate analysis to cells responding to challange in the FLIPR channel in the given frame range. This feature is off by default by the -1 value below. Range defining which frames to calculate ROI values. Frame number or from-to, or "All".
FLIPR response classifier min value (of range max, DfperF)	18	real	ROIs with greater df per f baseline-normalized FLIPR response than this value (baseline = 0) will be used only. The default -1 means that all ROIs are used.
Membrane potential calibration action	19	string	Select an action to be performed by the Membrane Potential Calibration Wizard
Calibration configuration file name (*.ips)	20	string	Configuration file previously saved from the Membrane Potential Calibration Wizard
Output Excel Data save file name (*.xlsx)	21	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 (aka PMPI, plasma membrane potential indicator; FLIPR plasma membrane potential assay kit) fluorescence time lapse recording to millivolts. In addition, post-hoc immunocytochemistry (ICC) images (showing the same view field after immunofluorescence staining) are used to selectively circle a specific cell type. 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 requires two Multi-Dimensional Open dialogs as input. One containing the live-cell time lapse of TMRM and FLIPR fluorescence, tagged as #1 in the top right corner of the dialog. The second dialog (tagged as #2) opens the post-hoc absorbance staining brioght field and a nuclear stain fluorescence images.
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 determine spectral crossbleed coefficients use the "Calculation of spectral unmixing coefficients for the ΔψM assay" interactive protocol in the Primer (see Help menu).
ROIs are generated automatically using the nuclear stain as seeds, following the shape of the maximum intensity projection of the summed FLIPR+TMRM image. Therefore if a cell moves during the experiment, the ROI will contain the cell for the whole duration of the recording. The calibration is not affected by the amount of background recorded in the ROI.

Gating ROIs to absorbance image:
* Input: Following the TMRM/FLIPR timelapse a stain e.g. dithizone for β-cell zinc content is applied to the cultures and the same view fields are recorded once. In addition dark current (no illumination) and blank (no cells, but the staining medium is present in an empty well) images are recorded as separate recordings. The blanks ideally recorded in multiple wells, so debris can be filtered out by median filtering below:
* First use the "Create background reference image(s) for multiwell plate using median of wells" pipeline from the Pipelines menu to process the dark current images. Alternatively an image can be also marked as Dark Current reference image from the context menu of the Image Window.
* Then use the "Create BLANK reference image for multiwell plate using median" pipeline from the Pipelines menu to process blank images. Alternatively an image can be also marked as Blank reference image from the context menu of the Image Window.
* The Dark Current and Blank images need to have the same channel number as the absorbance brightfield images opened in the Multi-Dimensional Open dialog #2. If not use the Edit / Rename in the main menu.
* During processing this pipeline, absorbance values will be calculated from the brightfield images, using the Dark Current and Blank images and only those ROIs are kept where the absorbance is higher than a threshold. The threshold is calculated based on a percentile of all ROIs, so at 50 percentile the most stained half of the ROIs will be kept.

Prepared TMRM and FLIPR images with immunofluorescence positive ROIs are passed to the Membrane Potential Calibration Wizard. 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.
This pipeline has been used in Gerencser in BBA Bioenergetics 2018 to gate recordings to pancreatic beta cells using dithizone staining.

Version history
V3
Function parameter rename: Minimum cell ICC fluorescence (%)
V4
INternal changes for Primer compatibility