diff --git a/Get_Linear_Unmixing_Parameters.groovy b/Get_Linear_Unmixing_Parameters.groovy
index e746755..7b74f34 100644
--- a/Get_Linear_Unmixing_Parameters.groovy
+++ b/Get_Linear_Unmixing_Parameters.groovy
@@ -1,180 +1,180 @@
/* = CODE DESCRIPTION =
* Implements linear unmixing as per the methods from
* Neher & Neher, J. Microsc. 213, 1 (2004), p. 46-62
* Zimmermann et al., FEBS Letters 531 (2992) p. 245-249
* Inspired by the ImageJ plugin from Joachim Walter
* https://imagej.nih.gov/ij/plugins/spectral-unmixing.html
*
*
* == INPUTS ==
* This script expect the following input folder configuration folder
* Main Folder
* - c1 [image name].tif
* - c2 [image name].tif
* - ...
* - ROI Sets
* - c1 [image name].tif.zip
* - c2 [image name].tif.zip
* - ...
*
* Each image is the individual control for channel 1, channel 2, etc...
*
* This configuration can be obtained using the BIOP MultiManual Select Tool
*
* === ABOUT THE ROI Sets ===
* There is no special name for the ROIs except one ROI per image that
* MUST contain the letters 'BG' so this script knows which ROIs are background
*
* Any other roi is considered as signal and the contribution of all ROIs will be averaged out
*
* Finally, you must provide a list of the fluorophore names, comma separated
*
* == OUTPUTS ==
* Running this code will produce an Umixing Parameters File to use with the
* 'Linear Unmixing.groovy' script
*
* = DEPENDENCIES =
* An up to date installation of Fiji should be enough to get this script to run
*
* = INSTALLATION =
* Simply open this script in the Fiji Script Interpreter and hit Run.
*
* = AUTHOR INFORMATION =
* Code written by Olivier Burri , EPFL - SV - PTECH - BIOP
* for Thierry Laroche, BIOP and Jessica Dessimoz, PTH
* September 13th 2018
*
* = COPYRIGHT =
* © All rights reserved. ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE, Switzerland, BioImaging And Optics Platform (BIOP), 2018
*
* Licensed under GNU General Public License (GLP) version 3
* This program is free software: you can redistribute it and/or modify
* it under the terms of the GNU General Public License as published by
* the Free Software Foundation, either version 3 of the License, or
* (at your option) any later version.
* This program is distributed in the hope that it will be useful,
* but WITHOUT ANY WARRANTY; without even the implied warranty of
* MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
* GNU General Public License for more details.
*
* You should have received a copy of the GNU General Public License
* along with this program. If not, see .
*/
#@File (label="Directory with Controls and ROIs", style="directory") image_dir
#@File (label="Output Unmixing Parameters File", style="save") output_file
#@String (label="Fluorophore Names, comma separated") fluorophore_names
import ij.*
import ij.plugin.frame.RoiManager
import ij.gui.Roi
import Jama.Matrix
import groovy.json.*
def mm = new MixingMatrixBuilder(fluorophore_names)
// Work on current image
// Get images and associated ROI Sets
def roi_dir = new File(image_dir, "ROI Sets")
println(image_dir)
// Iterate to get all the control images
image_dir.eachFile{ file ->
if (file.name.endsWith("tif")){
def image_file = file.name[(0..-5)]
def roi_file = new File(roi_dir, image_file+".zip")
def image = IJ.openImage(file.getAbsolutePath())
if( image != null && roi_file.exists() ) {
println ("Parsing Data for "+file.name)
// Open ROI Set
def rm = new RoiManager(false)
rm.runCommand("Open", roi_file.getAbsolutePath())
def rois = rm.getRoisAsArray() as List
// Images should start with c1, c2, etc so we can match which image is a control for which channel
- def channel = (file.name =~ /c(\d).*/).with { matches() ? it[0][1] as int : null }
+ def fluorophore = (file.name =~ /c(\d).*/).with { matches() ? it[0][1] as int : null }
// This measures the ROIs and stores them into the mixing array
- mm.measureIntensities(image, channel, rois)
+ mm.measureIntensities(image, fluorophore, rois)
}
}
}
// Save the arrays to JSON for further use
mm.saveToJson(output_file)
/**
* This class allows us to build the mixing matrix, the fluorophore names and the background
*/
class MixingMatrixBuilder {
int n_fluorophores
Matrix mixing_matrix
Matrix bg_matrix
def fluo_names
public MixingMatrixBuilder( String fluoro_names ) {
// Parse the fluorophore names and initialize the matrices we'll be needing
this.fluo_names = fluoro_names.tokenize(",").collect{it.trim()}
this.n_fluorophores = fluo_names.size()
this.mixing_matrix = new Matrix(n_fluorophores, n_fluorophores)
this.bg_matrix = new Matrix(n_fluorophores, n_fluorophores)
}
// This measures the BG and intensities for a single channel on a control image
- public measureIntensities(image, channel, rois) {
+ public measureIntensities(image, fluorophore, rois) {
// Find the BG ROI
Roi bg = rois.find{ it.getName().contains("BG") }
// Get all the ROIs that are NOT the BG
ArrayList others = rois.findAll{ !it.getName().contains("BG") }
// Now for all the channels of the image, get the intensities
- (1..n_fluorophores).each{fluorophore ->
+ (1..n_fluorophores).each{channel ->
// Set the right image processor to measure
- def proc = image.getStack().getProcessor(fluorophore)
+ def proc = image.getStack().getProcessor(channel)
proc.setRoi(bg)
// Measure the background
def bg_val = proc.getStatistics().mean
bg_matrix.set(channel-1, fluorophore-1, bg_val)
// Measure all the other ROIs
def val=0
others.each{roi ->
proc.setRoi(roi)
val += proc.getStatistics().mean
}
// Divide by number of ROIs to get average
val /= others.size()
// Final Mixing matrix value
mixing_matrix.set(channel-1, fluorophore-1, val)
}
mixing_matrix.print(6,3)
}
/*
* Saving a JSON object with
* the fluorophore names
* the background values for each image
* the mixing matrix
*/
public saveToJson(jsonFile) {
if (jsonFile.exists() ) jsonFile.delete()
def json = jsonFile << JsonOutput.toJson([fluo_names, bg_matrix, mixing_matrix])
}
}
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