CFP-YFP FRET - Fura crossbleed correction, FRET and Fura Ratio Image Calculation NFM

Parameters:

Name	#	Type	Description
Channel # of YFP	1	integer	The first linked image window with matching channel number will be invoked.
Channel # of CFP	2	integer	The first linked image window with matching channel number will be invoked.
Channel # of CFP-YFP FRET	3	integer	The first linked image window with matching channel number will be invoked.
Channel # of Fura 340nm	4	integer	The first linked image window with matching channel number will be invoked.
Channel # of Fura 380nm	5	integer	The first linked image window with matching channel number will be invoked.
Background Level (percentile)	6	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.

Description:

Subtracts background, measures crossbleed from Fura2 to the FRET construct and corrects spectral crossbleed and calculates 3-channel FRET image and Fura 340/380 image.
The Nonnegative Matrix Factorization requires spatially distinct objects labeled with each fluorophore. Therefore the crossbleed between CFP and YFP cannot be calculated with this technique, but the contribution of Fura (Ca2+-dependent) fluorescence can be, when the crossbleed factor calculation is constrained to cells that are bright with Fura but does not express the FRET probe. This is achieved by automated ROI drawing on the difference of the Fura380 and the YFP images. The "Starting Coefficient Matrix" of the "Blind Spectral Unmixing with NMF" encodes with zeros that no crossbleed is calculated between CFP, YFP and FRET channels and between Fura 340 and Fura 380 channels, but CFT (may) crossbleeds to Fura and Fura crossbleeds to all three FRET channels.
This is an example Pipeline, to be used with the FRET data set.
Based on the pipeline used in Gerencser et al. J Neurochem 2009:110 990-1004.

Version history
V2: tidied up structure