Plasma membrane potential and intracellular calcium single-cell analysis

Parameters:

Name	#	Type	Description
Channel Number for FLIPR	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 for Fluo	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.
Detector saturation level	3	real	Pixel intensity, frames with brighter maximal intensity than this will be ignored during segmentation, but not for plotting.
Debris cutoff for nucleus detection (percentile)	4	real	Brighter details of the image than this will be ignored. Decrease this value if bright debris results wrong scaling of nuclei.
Debris cutoff for cell shape detection (percentile)	5	real	Brighter details of the image than this will be ignored. Decrease this value if bright debris results wrong scaling of nuclei.
Approximate diameter of nuclei (holes in FLIPR fluorescence)	6	integer	Diameter of the nucleus in pixels
Minimum nucleus fluorescence (%)	7	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)	8	integer	Volume in 3D, area in 2D. 0 for not checking
Volume, maximum area (pixels)	9	integer	Volume in 3D, area in 2D. 0 for not checking
Minimum cell fluorescence (%)	10	real	Cells dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Background Level (Percentile)	11	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.
Save single cell fluorescence traces	12	boolean	Saves output as TAB delimited text file
Save file name for FLIPR (for txt file output)	13	string	See main menu Help/"Help on Expression Evaluation" for automatic file naming.
Save file name for Fluo (for txt file output)	14	string	See main menu Help/"Help on Expression Evaluation" for automatic file naming.
Membrane potential calibration action	15	string	Select an action to be performed by the Membrane Potential Calibration Wizard.
Calibration configuration file name (*.ips)	16	string	Configuration file saved from the Membrane Potential Calibration Wizard.
Output Excel Data save file name (*.xlsx)	17	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 calcium oscillation in insulinoma cells. The pipeline takes a two-channel confocal or wide field microscopic recording of FLIPR (Plasma Membrane Potential Indicator; Molecular Devices FLIPR Plasma membrane potential kit) and Fluo-3 or Fluo-4 time lapse. FLIPR typically does not stain nuclei, therefore single cells can be recognized from the hole in FLIPR fluorescence. The pipeline creates a single segmented image depicting the location of each identified cell and then measures FLIPR and Fluo fluorescence time courses over these segments. 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.
The algorithm was optimized for INS-1 832/13 cells recorded using a Zeiss LSM510 with Plan-Apochromat 40x/0.95 corr lens at 512x512 pixels and 0.44 µm/pixel resolution.

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 most cases (this pipeline does not include spectral unmixing).

Internal version: V12