The Use of FLIM-FRET for the Detection of Mitochondria-Associated Protein Interactions

Abstract

Fluorescence lifetime imaging microscopy–Förster resonant energy transfer (FLIM-FRET) is a high-resolution technique for the detection of protein interactions in live cells. As the cost of this technology becomes more competitive and methods are devised to extract more information from the FLIM images, this technique will be increasingly useful for studying protein interactions in live cells. Here we demonstrate the use of the ISS-Alba FLIM/FCS confocal microscope, which was custom-built for supervised automation of FLIM data acquisition. We provide a detailed protocol for collecting and analyzing good FLIM-FRET data. As an example, we use FLIM-FRET to detect the interaction between BclXL and Bad at the mitochondrial outer membrane in live MCF7 breast cancer cells.

Appendix

// macro to read in all the files and make them in a stack

dir1 = getDirectory("Choose tau Source Directory");

list1 = getFileList(dir1);

for (i=0; i<list1.length; i++) {

    open(dir1+list1[i]);

    nSlices;

    if (nSlices>1) {

        run("Stack to Images");

    }

}

run("Images to Stack", "name=tau title=t");

dir2 = getDirectory("Choose Ch1 Source Directory");

list2 = getFileList(dir2);

for (i=0; i<list2.length; i++) {

    open(dir2+list2[i]);

    nSlices;

    if (nSlices>1) {

        run("Stack to Images");

    }

}

run("Images to Stack", "name=Ch1 title=Ch1");

dir3 = getDirectory("Choose Ch2 Source Directory");

list3 = getFileList(dir3);

for (i=0; i<list3.length; i++) {

    open(dir3+list3[i]);

    nSlices;

    if (nSlices>1) {

        run("Stack to Images");

    }

}

run("Images to Stack", "name=Ch2 title=Ch2");

// to remove the images that have no cells

selectWindow("Ch2");

p=nSlices;

setBatchMode(true);

for (i=1; i<=p; i++) {

    setSlice(i);

    run("Measure");

    image_intensity=getResult("Mean");

    if (image_intensity<7) {

    run("Delete Slice");

    selectWindow("Ch1");

    setSlice(i);

    run("Delete Slice");

    selectWindow("tau");

    setSlice(i);

    run("Delete Slice");

    selectWindow("Ch2");

    i=i-1;

    p=p-1;

    }

}

// remove the bottom pixels (due to the problem of camera aligning) and resave single images into target folder

dir4 = getDirectory("Choose saving Directory");

selectWindow("Ch2");

n=nSlices;

for (i=1; i<=n; i++) {

    setSlice(i);

    run("Duplicate…", "title=Ch2-1");

    run("Canvas Size…", "width=256 height=250 position=Top-Center zero");

        saveAs("Tiff", dir4+"Ch2_"+i+".tif");

    close();

    }

selectWindow("Ch2");

close();

selectWindow("Ch1");

n=nSlices;

for (i=1; i<=n; i++) {

    setSlice(i);

    run("Duplicate…", "title=Ch1-1");

    run("Canvas Size…", "width=256 height=250 position=Top-Center zero");

        saveAs("Tiff", dir4+"Ch1_"+i+".tif");

    close();

    }

selectWindow("Ch1");

close();

selectWindow("tau");

n=nSlices;

for (i=1; i<=n; i++) {

    setSlice(i);

    run("Duplicate…", "title=tau-1");

    run("Canvas Size…", "width=256 height=250 position=Top-Center zero");

        saveAs("Tiff", dir4+"tau_"+i+".tif");

    close();

    }

selectWindow("tau");

close();

selectWindow("Results");

run("Clear Results");

//ROI selection for each image based on Ch2 signals and measure the intensities/lifetimes accordingly

for (i=1; i<=n; i++) {

    open(dir4+"Ch2_"+i+".tif");

    run("Duplicate…", "title=temp1.tif");

    selectWindow("temp1.tif");

    run("Subtract Background…", "rolling=50 stack");

    setAutoThreshold("Default dark");

    //run("Threshold…");

    getThreshold(lower, upper);

    new_lower=lower+50;

    setThreshold(new_lower, upper);

    run("Convert to Mask");

    run("Analyze Particles…", "size=8-Infinity pixel circularity=0.00-1.00 show=Nothing summarize add");

    selectWindow("Ch2_"+i+".tif");

    roiManager("Show None");

    roiManager("Show All");

    roiManager("Measure");

    q=nResults/2;

    o=0;

    for (m=0; m<q; m++) {

        r=m+q;

        max_intensity=getResult("Max", r);

        low_intensity=getResult("Min", r);

        if (max_intensity>1000 || (low_intensity<100 || low_intensity>400)) {

        w=m-o;

        roiManager("Select", w);

        roiManager("Delete");

        o=o+1;

        }

    }

    if (o<q) {

    roiManager("Save", dir4+"ROI"+i+".zip");

    run("Clear Results");

    selectWindow("Ch2_"+i+".tif");

    run("32-bit");

    run("Subtract Background…", "rolling=50 stack");

    roiManager("Show None");

    roiManager("Show All");

    roiManager("Measure");

    selectWindow("Results");

    saveAs("Results", dir4+"Ch2-"+i+".xls");

    run("Clear Results");

    open(dir4+"Ch1_"+i+".tif");

    run("32-bit");

    run("Subtract Background…", "rolling=50 stack");

    roiManager("Show None");

    roiManager("Show All");

    roiManager("Measure");

    selectWindow("Results");

    saveAs("Results", dir4+"Ch1-"+i+".xls");

    run("Clear Results");

    open(dir4+"tau_"+i+".tif");

    roiManager("Show None");

    roiManager("Show All");

    roiManager("Measure");

    selectWindow("Results");

    saveAs("Results", dir4+"tau-"+i+".xls");

    run("Clear Results");

    roiManager("reset");

    run("Close All");

    }

    else {

    run("Clear Results");

    roiManager("reset");

    run("Close All");

    }

}

run("Close All");

Dialog.create("Analysis is done.");

Dialog.show();