Spectral Unmix ( IAMatrixUnmix )
Parameters:
Name |
Short Name |
Type |
Description |
Coefficient Matrix |
coefficientmatrix |
string |
The matrix can be defined as Convolution kernels in the Preferences dialog, or define it here as ={{1,"probe 2 to channel 1",..},{"probe 1 to channel 2",1,..},....} |
Correct exposure time |
exposurecorrect |
boolean |
Set this Yes to normalize the cross-talk matrix with the exposure times |
Exposure correction for Coefficient Matrix |
expCorrMatrix |
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 |
expActual |
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:
Calculates unmixed fluorescence intensities by solving a linear equation with the cross-talk coefficient matrix.
Usage:
Subtract background before unmixing.
For two channels, where only one of the two channels bleeds into the other use the Tools/"Calculate Crossbleed Correction Factor" dialog or the "Calculate Simple Crossbleed" function to calculate Coefficient Matrix and automatically set its values into this function.
For mutual crossbleed between channels or for more than two channels use the "Blind Spectral Unmixing with NMF" function. That function also allows saving the calculated Coefficient Matrix as a named matrix or as an inline matrix for later use in this function.
Advanced use:
Coefficient matrix: each row corresponds to a channel, and tells that which fluorophores (columns) have how much contribution to this channel. The diagonal of the matrix contains ones for those channels which define a fluorophore.
The matrix can be defined as Convolution kernels in the Preferences dialog, or optionally define crossbleed as inline matrix ={{1,"probe 2 to channel 1",..},{"probe 1 to channel 2",1,..},....}.
There may be fewer fluorophores than channels, in this case some of the columns contains only zeros.
If exposure times have been disproportionally changed since calculating the Coefficient Matrix, us exposure correction below.
Exposure correction vectors: declared as row or column kernels; containing the exposure times for each channel.
Exposure correction for Coefficient Matrix: exposure times used for acquiring images for coefficient calculation.
Exposure correction for Image data: exposure times used for acquiring images to be corrected.
Exposure correction matrices can be entered here as ={{a,b,c,...}} where a,b,c are exposure times in identical units.
Copy/paste matrices from Excel or from TSV/CSV files: copy the matrix from Excel, paste into the Coefficient Matrix field, press enter and the down arrow to display the converted matrix.