Image processing pipelines in Image Analyst MKII
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CFP-YFP FRET - Fura crossbleed correction, FRET and Fura Ratio Image Calculation

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.
Spectral Unmixing 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.
Correct exposure time 8 boolean Set this True to normalize the cross-talk matrix with the exposure times
Exposure correction for Coefficient Matrix 9 string A column or row vector can be defined as Convolution kernels in the Preferences dialog, or define it here as ={{a,b,c,...}}
Exposure correction for Image data 10 string A column or row vector can be defined as Convolution kernels in the Preferences dialog, or define it here as ={{a,b,c,...}}
Description:
Subtracts background, corrects spectral crossbleed and calculates 3-channel FRET image and Fura 340/380 ratio image.
Crossbleed was separately calculated using separate CFP, YFP and Fura samples.
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.