pixFRET - running the plugin for time lapse images // looping?

Question

I just recently started to work with ImageJ (and thus do not have much experience with macro programming) to analyze my microscopy pictures. In order to generate FRET pixel-by-pixel images that are corrected for spectral bleed through I am using the plug in: pixFRET. This plug in requires a stack of 3 images to work: FRET, Donor, Acceptor. So far, I have to open every picture myself and this is REALLY inconvenient for large time stacks (> 1000 images). I am looking for a way to loop the plug in or create some kind of macro to do this.

A short description of my Data structure: workfolder\filename_t001c1 (Channel 1 Image - Donor at time point 001), filename_t001c2 (Channel 2 Image - FRET at time point 001), ...t001c3 (can be neglected) ...t001c4 (Channel 4 Image - Acceptor at time point 001).

I would have to create a stack of C2/C1/C4 at each time point that is automatically analyzed by pixFRET (with set parameters) and the result should be saved in an output folder.

I am grateful for every suggestion as my biggest problem is the looping of this whole stack generation/pixFRET analysis (can only do this manual right now).

Thanks David

Answer 1

I did not find a way to directly include the parameters and commands from the pixFRET PlugIn. However, here I show a work around that works with IJ_Robot to add these commands. I further included some stuff to perform a alignment of the camera channels based on the first images of the time series.

   // Macro for creating time resolved pixFRET images with a alignment of both cameras used
// a separate setting file is required for pixFRET -> put this into the same folder as the pixFRET plugin
// the background region has to be set manually in this macro
// IJ_robot uses cursor movements - DO NOT move the cursor while excuting the macro + adjust IJ_robot coordinates when changing the resolution/system.




dir = getDirectory("Select Directory");
list = getFileList(dir);

//single alignment
 run("Image Sequence...", "open=[dir] number=2 starting=1 increment=1 scale=100 file=[] or=[] sort");
rename(File.getName(dir));
WindowTitle=getTitle()
rename(WindowTitle+toString(" Main"))
MainWindow=getTitle()
NSlices=getSliceNumber()
xValue=getWidth()/2
yValue=getHeight()/2

//setTool("rectangle");
makeRectangle(0, 0, xValue, yValue);
run("Align slices in stack...", "method=5 windowsizex="+toString(xValue*2-20)+" windowsizey="+toString(yValue*2-20)+" x0=10 y0=10 swindow=0 ref.slice=1 show=true");
selectWindow("Results");

XShift=getResult("dX", 0);
YShift=getResult("dY", 0);


File.makeDirectory(toString(File.getParent(dir))+toString("\\")+"test"+" FRET");

for(i=0;i<list.length;i+=4){
open(dir+list[i+1]);
run("Translate...", "x=XShift y=YShift interpolation=None stack");

open(dir+list[i]);


open(dir+list[i+3]);
run("Translate...", "x=XShift y=YShift interpolation=None stack");

wait(1000);
run("Images to Stack", "name=Stack title=[] use");
selectWindow("Stack");
makeRectangle(15, 147, 82, 75); //background region
run("PixFRET...");
run("IJ Robot", "order=Left_Click x_point=886 y_point=321 delay=500 keypress=[]");
run("IJ Robot", "order=Left_Click x_point=874 y_point=557 delay=500 keypress=[]");
selectWindow("NFRET (x100) of Stack");

save(toString(File.getParent(dir))+toString("\\")+"test"+" FRET"+toString(i) +".tif");

selectWindow("Stack");
close();
selectWindow("FRET of Stack");
close();

selectWindow("NFRET (x100) of Stack");
close();
run("IJ Robot", "order=Left_Click x_point=941 y_point=57 delay=300 keypress=[]");
}

Thanks for your help Jan. If you can think of a way to call these pixFRET commands directly rather than using Ij_robot, please let me know.

Answer 2

Take this tutorial from Fiji (is just ImageJ) as a starting point, and use the macro recorder (Plugins > Macros > Record...) to get the neccessary commands.

Your macro code could then look something like this:

function pixfret(path, commonfilename) {
    open(path + commonfilename + "c2");
    open(path + commonfilename + "c1");
    open(path + commonfilename + "c4");
    run("Images to Stack", "name=Stack title=[] use");
    run("PixFRET"); // please adjust this to your needs
}

setBatchMode(true); 
n_timepoints = 999;
dir = "/path/to/your/images/";
for (i = 0; i < n_timepoints; i++)
    pixfret(dir, "filename_t" + IJ.pad(i, 4));
setBatchMode(false);

Hope that helps.