Calculate Simple Crossbleed ( IASimpleCrossbleed )

Parameters:

Name	Short Name	Type	Description
Direction of crossbleed between two channels	direction	string	Which channel bleeds into the other in the order of selection as Image A. Available directions: "first->second","second->first"
Use frame #s (0 is All)	frame	integer	Set which frame to use for the calculation or use 0 to use frames.
Coefficient matrix to save	matrix	string	Matrix name to save crossbleed matrix
Set this matrix to Spectral Unmix function	send	boolean	The above matrix name will be the default in the "Spectral Unmix" function.
Min intensity (% relative to max percentile)	pmin	real	Details brighter than this percent intensity (calculated between 0.1 and 99.9 percentiles) of the crossbleeding image series will be used for the calculation.
Max percentile (debris cutoff)	pmax	real	Details brighter than this percentile in either channel will no be used for calculations.
Constrain to ROIs (union of all)	constrainActiveROI	boolean	Unmixing coefficients are calculated from the pixels of all ROIs.
Channel of fluorophore	sourcechannel	integer	For the "one fluorophore to multiple channels" direction set here to which channel to assign the fluorphore.

Description:

Calculation of the amount of spectral crossbleed from one channel to the other. All frames of the time series or the indicated one (Use frame#) is used for the calculation. Use this simple calculation to unmix two channels, when only one channel bleeds into the other but there is no crossbleed into the opposite direction. The calculation is identical to the one in the Tools/"Calculate Crossbleed Correction Factor" dialog. If both channels bleed into the other use single-probe stained images as given below at Usage to calculate a factor for each. Alternatively, for mutual crossbleed between channels or for more than two channels use the "Blind Spectral Unmixing with NMF" function to calculate crossbleed without the requirement for single-probe stained recordings.
Direction: Set which channel bleeds into the other in the order as images were selected as Image A.
Coefficient matrix to save: the result will be saved as a matrix listed in the Edit/Preferences/"Convolution kernels, Matrices", and available for use in the "Spectral Unmix" function.
Set this matrix to Spectral Unmix function: enable this to make the above matrix name default when you switch over to "Spectral Unmix".
Usage:
Calculate coefficients on a pair of control images, e.g. single fluorophore stained samples using this function, and then unmix other images using the "Spectral Unmix" function.
If both channels bleed into the other and a pair of single-probe stained two-channel recordings are available, calculate the crossbleed using this function for both recordings and then merge the top right and bottom left coefficients into a single matrix.
"one fluorophore to multiple channels" direction: calculates crossbleed of a single fluorophore to multiple channels. Select all channels as imput, and set "Channel of fluorophore" to the channel number, where to assign the present fluorophore. Use this approach for calculating larger crossbleed matrices using single-stains. The matrix is created in the Excel Data Window.
The output is the first input.