Prepare two-channel time lapse for intensity analysis with spectral unmixing and subtraction of inhomogeneous background

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.
Subtract reference image background	3	boolean	Yes executes output #1, No executes output #2.
Background level (percentile)	4	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.
Local background subtraction by median rolling ball	5	boolean	Yes executes output #1, No executes output #2.
Median rolling ball size	6	integer	The median is calculated over a width x width rectangular area around each pixel of the image.
Spectral Unmix Coefficient Matrix	7	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	8	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)	9	integer	1 if comparing to the first frame. Use more frames if not comparing to the first frame and the view field drifts slowly.

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). To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used. In addition to this, inhomogeneous background can be removed by image or by local background subtraction.

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 FLIPR).
This pipeline is suggested to be used with the Membrane Potential Calibration Wizard for mitochondrial membrane potential measurements.

Version history:
V2
Inhomogeneous background subtraction options have been added.