Prepare two channel time lapse for intensity analysis with LOCAL background subtraction and spectral unmixing

Parameters:

Name	#	Type	Description
Channel number #1	1	integer	The first linked image window with matching channel number will be invoked.
Channel number #2	2	integer	The first linked image window with matching channel number will be invoked.
Background level (percentile)	3	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	4	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	5	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
Image stabilizer period (frames)	6	integer	1 if comparing to first frame. Use more frames if not comparing to first frame and the view filed moves slowly.
Median filter size (pixels)	7	integer	A copy of the original image is median filtered with this window size, and subtracted from the original image.

Description:

This 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). In addition, local background is subtracted. Local background subtraction is efficient if cells are very small, and the background is locally inhomogeneous. However local background subtraction may introduce artifacts if brightness of nearby objects changes therefore it is to be used with caution.
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.
Use: generic pre-processing for two-channel recordings, before single-cell analysis. Applicable to Ca2+-imaging (e.g. Fura-2, yellow cameleons), other ratiometric ion probes imaging (e.g. PBFI, SPFI, SNARF-1, BCECF), reactive oxygen species imaging (e.g. HyPer, roGFP), intramolecular FRET sensor imaging, potentiometric probes (TMRM plus PMPI).
This pipeline is suggested to be used with the Membrane Potential Calibration Wizard for mitochondrial membrane potential measurements as an alternative to the pipeline “Prepare two channel time lapse for intensity analysis with spectral unmixing”.