Plasma membrane potential and GFP intensity single-cell analysis using post-hoc seed image

Parameters:

Name	#	Type	Description
Channel Number for FLIPR (cell shape is determined from this; (Multi-Dimensional Open #1)	1	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog #1. Use Edit/Rename to view or change channel number.
Channel Number for GFP-based probe (Multi-Dimensional Open #1)	2	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog #1. Use Edit/Rename to view or change channel number.
Channel Number for post-hoc nuclear marker (Multi-Dimensional Open #2)	3	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog #2. Use Edit/Rename to view or change channel number.
Background Level (Percentile)	4	real	Background is calculated as frame-by-frame mean of those pixels that are the darker than this percentile of image histogram for the duration of the entire recording.
Spectral Unmix Coefficient Matrix	5	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.
Spectral Unmix: correct exposure time	6	boolean	Set this Yes to normalize the cross-talk matrix with the exposure times
Spectral Unmix: Exposure correction for Coefficient Matrix	7	string	A column or row vector can be defined as Convolution kernels in the Preferences dialog, or define it here as ={{a,b,c,...}}
Spectral Unmix: Exposure correction for Image data	8	string	A column or row vector can be defined as Convolution kernels in the Preferences dialog, or define it here as ={{a,b,c,...}}
Channel alignment based on only the first frame	9	boolean	Calculates shift from first few frames, then aligns the whole time lapse. No aligns each frame of the time lapse. The number of frames is set by the value used for ΔF/F0 averaging in the Preferences
Image stabilizer compares to first frame	10	boolean	This 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 to the first frame any more.
Image stabilizer period (frames)	11	integer	1 if comparing to first frame. Use more frames if not comparing to first frame and the view filed moves slowly.
Debris cutoff for nucleus detection (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".
Debris cutoff for cell shape detection (percentile)	13	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".
Approximate diameter of nuclei	14	integer	Diameter of the nucleus in pixels
Minimum nucleus fluorescence (%)	15	real	Cells dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Cell size, minimum area (pixels)	16	integer	Volume in 3D, area in 2D. 0 for not checking
Cell size, maximum area (pixels)	17	integer	Volume in 3D, area in 2D. 0 for not checking
Minimum cell fluorescence (%)	18	real	Cells dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Save single cell fluorescence traces	19	boolean	Saves output as TAB delimited text file
Save file name for probe #1 (for txt file output)	20	string	See main menu Help/"Help on Expression Evaluation" for automatic file naming.
Save file name for probe #2 (for txt file output)	21	string	See main menu Help/"Help on Expression Evaluation" for automatic file naming.
Membrane potential calibration action	22	string	Select an action to be performed by the Membrane Potential Calibration Wizard
Calibration configuration file name (*.ips)	23	string	Configuration file saved from the Membrane Potential Calibration Wizard
Output Excel Data save file name (*.xlsx)	24	string	Excel Data window output will be created and saved using this name.

Description:

This pipeline was originally used for single cell analysis of plasma membrane potential and insulin exocytosis using superecliptic synaptopHluorin in insulinoma cells. The pipeline analyzes a two-channel fluorescence (e.g. FLIPR and a GFP-based probe, such as synaptopHluorin) time-lapse recording on the single-cell level, using a post-hoc nuclear stain. For the post-hoc recording the same view field is captured with the addition of a nuclear stain e.g. Hoechst 33342 after the functional time lapse has been concluded (this is an additional channel to the two probe-channels during live imaging). The pipeline loads the functional time lapse from Multi-Dimensional Open #1 (tag the dialog in the top right corner) and the post-hoc stain image form Multi-Dimensional Open #2. The pipeline creates a single segmented image depicting the location of each identified cell based on the nuclei and the FLIPR fluorescence. Fluorescence intensities are measured in stabilized, background subtracted and spectrally unmixed images of the original probe fluorescence. The output is saved as text files (or alternatively can be entered into the Excel Data Window. At the end the Membrane Potential Calibration Wizard is called, so potentials can be calculated from FLIPR fluorescence traces.

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. This pipeline also allows for using different exposure times during measurement of spectral crossbleed coefficients and during the actual experiment, that is analyzed by this pipeline.

The pipeline is applicable to combination of FLIPR with any spectrally distinguishable single wavelength probe, including GFP-based probes, green-fluorescent calcium indicators Fluo-3 and Fluo-4, or red-fluorescent rhodamine derivatives such as Rhod-2. Spectral cross-bleed has to be corrected in each case.

Internal version: V11