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2021-03-05-BatchSTORM_Hadjer_edited.ijm
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2021-03-05-BatchSTORM_Hadjer_edited.ijm
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// This macro batch-analyzes PALM data
macro "Batch Process STORM data " { convert("Raw Data"); }
function convert(format) {
requires("1.33s");
// Default values for the Options Panel
SR_SIZE_DEF = 16;
//ADU = 4.86 // Add real ADU 10MHz emccd gain 3
// ADU= 1.4 - 3MHX conv gain3
//ADU=5.5 17MHz emccd
// Titles of the Thunderstorm windows for catching them
RESULTS_TITLE = "ThunderSTORM: results";
//RECON_TITLE = "Normalized Gaussian";
RECON_TITLE = "Averaged shifted histograms";
title = "Untitled";
width=512;
height=512;
Dialog.create("Test");
Dialog.addString("Directory:", "C:\_data\_storm data");
Dialog.addCheckbox("Save .csv", true);
Dialog.addCheckbox("STD", true);
Dialog.addCheckbox("QuickPALM", false);
Dialog.addCheckbox("DoM ", true);
Dialog.addCheckbox("ThunderSTORM (Phasor)", true);
Dialog.addCheckbox("ThunderSTORM (Gaussia)", false);
Dialog.addCheckbox("Split frames odd/even", false);
Dialog.addMessage("Camera Options \n") ;
Dialog.addNumber("Pixel Size", 144);
Dialog.addNumber("Intensity Threshold", 4);
Dialog.addNumber("ADU", 1.4);
Dialog.addNumber("Noise",1);
Dialog.addNumber("BASELINE",200);
Dialog.addMessage("Post-processing") ;
Dialog.addNumber("Detection Threshold (AU)",300);
Dialog.addNumber("THRESHOLD (photon)",100);
Dialog.addNumber("GROUPING_RADIUS (nm)",100);
Dialog.addMessage("Reconstruction Settings") ;
Dialog.addNumber("SR_Pixel Size (nm)",10);
Dialog.show();
directory = Dialog.getString();
savecsv = Dialog.getCheckbox();
doSTD = Dialog.getCheckbox();
doQuickPALM = Dialog.getCheckbox();
doDoM = Dialog.getCheckbox();
doTSP= Dialog.getCheckbox();
doTSG = Dialog.getCheckbox();
doSplit = Dialog.getCheckbox();
PIXEL_SIZE = Dialog.getNumber();
intensity_threshold = Dialog.getNumber();
sigma=150/PIXEL_SIZE; //do better than that
ADU = Dialog.getNumber();
NOISE= Dialog.getNumber();
BASELINE= Dialog.getNumber();
Det_THRESH= Dialog.getNumber();
THRESH= Dialog.getNumber();
GROUPING_RADIUS = Dialog.getNumber();
SRPIXEL = Dialog.getNumber();
MAG=PIXEL_SIZE/SRPIXEL;
//add backslash if missing in dir name
if (endsWith(directory,"\ " ))
{}
else
{
directory=directory+File.separator;
}
dir1=directory;
// read in file listing from source directory
list = getFileList(dir1);
// loop over the files in the source directory
setBatchMode(true);
for (i=0; i<list.length; i++) {
showProgress(i+1, list.length);
if (!endsWith(list[i], '.tif')){
print("Not TIF: "+dir1+list[i]);
}
else {
open(dir1+list[i]);
name= list[i];
index = lastIndexOf(name, ".");
name = substring(name, 0, index);
numstack=nSlices();
if(numstack>100)
{
rename("tmp");
run("Add...", "value = 400");
//Splitting the frames into odd and even frames and calculating the FRC
if (doSplit==true) {
dir_even = dir1+"/"+ name +"Even/";
File.makeDirectory(dir_even);
dir_odd = dir1+"/"+ name + "Odd/";
File.makeDirectory(dir_odd);
dir_even_FRC = dir_even+"/FRC/";
File.makeDirectory(dir_even_FRC);
dir_odd_FRC = dir_odd+"/FRC/";
File.makeDirectory(dir_odd_FRC);
run("Split image sequence into odd and even frames");
rename("image");
run("Deinterleave", "how=2");
selectWindow("image #1");
saveAs("TIF", dir_odd+name+"\.tif");
if (doDoM==true) {
run("Detect Molecules", "task=[Detect molecules and fit] psf="+sigma+ " intensity=" + intensity_threshold + " pixel="+PIXEL_SIZE+" parallel=1000 fitting=5 ignore");
//if (numstack>4000)
//{
//run("Drift Correction", "pixel=20 batch=2000 method=[Direct CC first batch and all others] apply");
//}
w=getWidth();
h=getHeight();
run("Reconstruct Image", "for=[Only true positives] pixel="+SRPIXEL+" width="+w+" height="+h+" sd=[Constant value] value=10 cut=25 x_offset=0 y_offset=0 range=1-99999 render=Z-stack z-distance=100 lut=Fire");
selectWindow("Reconstructed Image");
saveAs("Tiff", dir_odd+name+"_DOM.tif");
saveAs("Tiff", dir_odd_FRC+name+"_DOM.tif");
saveAs("Results",dir_odd+name+"_DOM.xls");
close();
};
if (numstack>3000)
{
run("Drift Correction", "pixel=20 batch=500 method=[Direct CC first batch and all others] apply");
run("Reconstruct Image", "for=[Only true positives] pixel="+SRPIXEL+" width="+w+" height="+h+" sd=[Constant value] value=10 cut=25 x_offset=0 y_offset=0 range=1-99999 update render=Z-stack z-distance=100 lut=Fire");
selectWindow("Reconstructed Image");
saveAs("Tiff", dir_odd+name+"_DOM_dedrift.tif");
saveAs("Tiff", dir_odd_FRC+name+"_DOM_dedrift.tif");
saveAs("Results",dir_odd+name+"_DOM_dedrift.xls");
close();
close("Drift Correction (frames=1500 px size=20 nm)");
run("Drift Correction", "pixel=20 batch=500 method=[Direct CC first batch and all others] apply");
run("Reconstruct Image", "for=[Only true positives] pixel="+SRPIXEL+" width="+w+" height="+h+" sd=[Constant value] value=10 cut=25 x_offset=0 y_offset=0 range=1-99999 average update render=Z-stack z-distance=100 lut=Fire");
selectWindow("Reconstructed Image");
saveAs("Tiff", dir_odd+name+"_DOM_dedrift_avg.tif");
saveAs("Tiff", dir_odd_FRC+name+"_DOM_dedrift_avg.tif");
saveAs("Results",dir_odd+name+"_DOM_dedrift_avg.xls");
close();
close("Drift Correction (frames=1500 px size=20 nm)");
}
rename("tmp");
selectWindow("image #2");
saveAs("TIF", dir_even+name+"\.tif");
if (doDoM==true) {
run("Detect Molecules", "task=[Detect molecules and fit] psf="+sigma+ " intensity=" + intensity_threshold + " pixel="+ PIXEL_SIZE +" parallel=1000 fitting=5 ignore");
//if (numstack>4000)
//{
//run("Drift Correction", "pixel=20 batch=2000 method=[Direct CC first batch and all others] apply");
//}
w=getWidth();
h=getHeight();
run("Reconstruct Image", "for=[Only true positives] pixel="+SRPIXEL+" width="+w+" height="+h+" sd=[Constant value] value=10 cut=25 x_offset=0 y_offset=0 range=1-99999 render=Z-stack z-distance=100 lut=Fire");
selectWindow("Reconstructed Image");
saveAs("Tiff", dir_even+name+"_DOM.tif");
saveAs("Tiff", dir_even_FRC+name+"_DOM.tif");
saveAs("Results",dir_even+name+"_DOM.xls");
close();
};
if (numstack>3000)
{
run("Drift Correction", "pixel=20 batch=500 method=[Direct CC first batch and all others] apply");
run("Reconstruct Image", "for=[Only true positives] pixel="+SRPIXEL+" width="+w+" height="+h+" sd=[Constant value] value=10 cut=25 x_offset=0 y_offset=0 range=1-99999 update render=Z-stack z-distance=100 lut=Fire");
selectWindow("Reconstructed Image");
saveAs("Tiff", dir_even+name+"_DOM_dedrift.tif");
saveAs("Tiff", dir_even_FRC+name+"_DOM_dedrift.tif");
saveAs("Results",dir_even+name+"_DOM_dedrift.xls");
close();
close("Drift Correction (frames=1500 px size=20 nm)");
run("Drift Correction", "pixel=20 batch=500 method=[Direct CC first batch and all others] apply");
run("Reconstruct Image", "for=[Only true positives] pixel="+SRPIXEL+" width="+w+" height="+h+" sd=[Constant value] value=10 cut=25 x_offset=0 y_offset=0 range=1-99999 average update render=Z-stack z-distance=100 lut=Fire");
selectWindow("Reconstructed Image");
saveAs("Tiff", dir_even+name+"_DOM_dedrift_avg.tif");
saveAs("Tiff", dir_even_FRC+name+"_DOM_dedrift_avg.tif");
saveAs("Results",dir_even+name+"_DOM_dedrift_avg.xls");
close();
close("Drift Correction (frames=1500 px size=20 nm)");
}
run("FRC Calculation (Batch)...", "first=[" + dir_even_FRC + "] second=[" + dir_odd_FRC + "] resolution=[Fixed 1/7]");
selectWindow("FRC Results");
saveAs("Results", dir1+name+"FRC.xls");
close();
}
selectWindow("tmp");
//STD
if (doSTD==true) {
run("Z Project...", "projection=[Standard Deviation]");
run("Scale...", "x=2 y=2 interpolation=Bilinear create");
saveAs("PNG", dir1+name+"_std.png");
close();
close();
}
selectWindow("tmp");
w=getWidth();
h=getHeight();
//DoM
if (doDoM==true) {
run("Detect Molecules", "task=[Detect molecules and fit] psf="+sigma+ " intensity=" + intensity_threshold + " pixel="+PIXEL_SIZE+" parallel=1000 fitting=5 ignore");
//if (numstack>4000)
//{
//run("Drift Correction", "pixel=20 batch=2000 method=[Direct CC first batch and all others] apply");
//}
run("Reconstruct Image", "for=[Only true positives] pixel="+SRPIXEL+" width="+w+" height="+h+" sd=[Constant value] value=10 cut=25 x_offset=0 y_offset=0 range=1-99999 render=Z-stack z-distance=100 lut=Fire");
selectWindow("Reconstructed Image");
saveAs("Tiff", dir1+name+"_DOM.tif");
saveAs("Results",dir1+name+"_DOM.xls");
close();
if (numstack>3000)
{
run("Drift Correction", "pixel=20 batch=1500 method=[Direct CC first batch and all others] apply");
run("Reconstruct Image", "for=[Only true positives] pixel="+SRPIXEL+" width="+w+" height="+h+" sd=[Constant value] value=10 cut=25 x_offset=0 y_offset=0 range=1-99999 update render=Z-stack z-distance=100 lut=Fire");
selectWindow("Reconstructed Image");
saveAs("Tiff", dir1+name+"_DOM_dedrift.tif");
saveAs("Results",dir1+name+"_DOM_dedrift.xls");
close();
close("Drift Correction (frames=1500 px size=20 nm)");
run("Drift Correction", "pixel=20 batch=1500 method=[Direct CC first batch and all others] apply");
run("Reconstruct Image", "for=[Only true positives] pixel="+SRPIXEL+" width="+w+" height="+h+" sd=[Constant value] value=10 cut=25 x_offset=0 y_offset=0 range=1-99999 average update render=Z-stack z-distance=100 lut=Fire");
selectWindow("Reconstructed Image");
saveAs("Tiff", dir1+name+"_DOM_dedrift_avg.tif");
saveAs("Results",dir1+name+"_DOM_dedrift_avg.xls");
close();
close("Drift Correction (frames=1500 px size=20 nm)");
}
}
//TSP
if (doTSP==true) {
run("Camera setup", "isemgain=false pixelsize=" + PIXEL_SIZE + " photons2adu=" + ADU +" quantumefficiency=0.89 offset="+BASELINE+" readoutnoise=" + NOISE);
selectWindow("tmp");
run("Run analysis", "filter=[Wavelet filter (B-Spline)] scale=2.0 order=3 detector=[Centroid of connected components] watershed=true threshold=std(Wave.F1) estimator=[Phasor-based localisation 2D] astigmatism=false fitradius=2 calibrationpath= renderer=[Averaged shifted histograms] magnification="+MAG+" colorize=false threed=false shifts=2 repaint=500");
run("Show results table", "action=filter formula=intensity>"+THRESH);
//run("Show results table", "action=density neighbors=5 radius=50.0 dimensions=2D");
run("Show results table", "action=drift magnification=5.0 method=[Cross correlation] ccsmoothingbandwidth=0.25 save=false steps=10 showcorrelations=false");
selectWindow(RECON_TITLE);
saveAs("Tiff", dir1+name+"_TS_P.tif");
close();
selectWindow(RESULTS_TITLE);
run("Export results", "floatprecision=3 filepath=["+dir1+name+"_TS_P.csv] fileformat=[CSV (comma separated)] intensity=true offset=true saveprotocol=false x=true sigma2=true y=true sigma1=true z=true bkgstd=true id=true frame=true");
// run("Show results table", "action=merge zcoordweight=0.1 offframes=1 dist="+GROUPING_RADIUS+" framespermolecule=0");
// run("Export results", "floatprecision=3 filepath=["+dir1+name+"_TS_P_GP.csv] fileformat=[CSV (comma separated)] intensity=true offset=true saveprotocol=false x=true sigma2=true y=true sigma1=true z=true bkgstd=true id=true frame=true");
close();
}
//TSG
if (doTSG==true) {
run("Camera setup", "isemgain=false pixelsize=" + PIXEL_SIZE + " photons2adu=" + ADU +" quantumefficiency=0.89 offset="+BASELINE+" readoutnoise=" + NOISE);
selectWindow("tmp");
run("Run analysis", "filter=[Wavelet filter (B-Spline)] scale=2.0 order=3 detector=[Centroid of connected components] watershed=true threshold=std(Wave.F1) estimator=[PSF: Integrated Gaussian] sigma="+sigma+" fitradius=3 method=[Weighted Least squares] full_image_fitting=false mfaenabled=false renderer=[Averaged shifted histograms] magnification="+MAG+" colorize=false threed=false shifts=2 repaint=500");
run("Show results table", "action=filter formula=intensity>"+THRESH);
run("Show results table", "action=density neighbors=5 radius=50.0 dimensions=2D");
run("Show results table", "action=drift magnification=5.0 method=[Cross correlation] ccsmoothingbandwidth=0.25 save=false steps=10 showcorrelations=false");
selectWindow(RECON_TITLE);
saveAs("Tiff", dir1+name+"_TS_G.tif");
close();
selectWindow(RESULTS_TITLE);
run("Export results", "floatprecision=3 filepath=["+dir1+name+"_TS_G.csv] fileformat=[CSV (comma separated)] sigma=true intensity=true chi2=true offset=true saveprotocol=true x=true y=true bkgstd=true id=true uncertainty_xy=true frame=true");
run("Show results table", "action=merge zcoordweight=0.1 offframes=1 dist="+GROUPING_RADIUS+" framespermolecule=0");
run("Export results", "floatprecision=3 filepath=["+dir1+name+"_TS_G_GP.csv] fileformat=[CSV (comma separated)] sigma=true intensity=true chi2=true offset=true saveprotocol=true x=true y=true bkgstd=true id=true uncertainty_xy=true frame=true detections=true");
close();
}
//QuickPALM
if (doQuickPALM==true) {
selectWindow("tmp");
run("Analyse Particles", "minimum=3 maximum=4 image=160 smart online stream file=D:\\test.xls pixel=30 accumulate=0 update=100 _image=imgNNNNNNNNN.tif start=0 in=50 _minimum=50 local=20 _maximum=1000 threads=50");
run("16-bit");
run("Multiply...", "value=3.000");
run("Smooth");
run("Scale Bar...", "width=1000 height=4 font=14 color=White background=None location=[Lower Right] bold");
saveAs("Tiff", dir1+name+"_QkPALM.tif");
close();
close();
open("D:\\test.xls");
saveAs("Results",dir1+name+".xls");
}
run("Close All");
close("Drift*");
close("*.csv");
close("\\Others");
close("Histo*");
close("*");
}
else
close();
}
//run("ThunderSTORM export", "results=ThunderSTORM photoelectrons=1.4 base=100 value=100 export=["+ dir1+name+"_TS_G.xls]");
};
run("Dispose All Windows", "/all image non-image");
}