Hi,

I open an 32bit image from our image database, which is the result of a ratiomatric anlysis. Areas with cells contains values >0, but during the ratio calculation there was an threshold, so that certain areas inside the image will be set to zero.

Inside Fiji I drew some ROIs, which I usually plot over time and to do so I wrote macro, which cycles through all the frames, calulates the mean of every ROIs and plots them. Unfortunately this does not work, when all pixels inside one ROI (for instance a background ROI) are zero.

I also create a new images with 10 frames and all zeros inside from File --> New, drew an ROI and started the macro. The same error message came up.


java.lang.NullPointerException: null
	 at ij.plugin.filter.Analyzer.saveResults(Analyzer.java:409)
	 at ij.plugin.filter.Analyzer.measure(Analyzer.java:241)
	 at ij.plugin.frame.RoiManager.multiMeasure(RoiManager.java:882)
	 at ij.plugin.frame.RoiManager.runCommand(RoiManager.java:1612)
	 at ij.macro.Functions.roiManager(Functions.java:2400)
	 at ij.macro.Functions.getFunctionValue(Functions.java:215)
	 at ij.macro.Interpreter.getFactor(Interpreter.java:1296)
	 at ij.macro.Interpreter.getTerm(Interpreter.java:1267)
	 at ij.macro.Interpreter.getStringExpression(Interpreter.java:1403)
	 at ij.macro.Interpreter.getStringTerm(Interpreter.java:1226)
	 at ij.macro.Interpreter.getString(Interpreter.java:1183)
	 at ij.macro.Interpreter.doStatement(Interpreter.java:259)
	 at ij.macro.Interpreter.doStatements(Interpreter.java:202)
	 at ij.macro.Interpreter.run(Interpreter.java:103)
	 at ij.macro.Interpreter.run(Interpreter.java:73)
	 at CLI.Refresh_Macros.runScript(Refresh_Macros.java:56)
	 at common.RefreshScripts.runScript(RefreshScripts.java:325)
	 at fiji.scripting.TextEditor$Tab$6.execute(TextEditor.java:1216)
	 at fiji.scripting.TextEditor$Executer$1.run(TextEditor.java:1776)

When I addes 0.0001 to the complete image, the error was gone. So it looks to me like the mean value calulation fails under certain circumstances. I also tries to change Misc Option - Divide by zero value from inifinity to 0.001, but that did not help.

Here is the macro code, if you want to try it out.



// Author: Sebastian Rhode
// Date: 2011-04-26

version_number = 1.6;

// marco for plotting mutiple lines from up-to 8 ROIs inside one plot
// only works with time-lapse images
// does not work with image 5D format

// get title from active window
title_orig = getTitle();
selectWindow(title_orig);
imagedir = getInfo("image.directory")
imagefilename = getInfo("image.filename")
//print(imagedir);
print(imagedir + imagefilename);
// get dimension from original image
getDimensions(w,h,ch,sl,fr);
print("X :",w,"Y :",h,"CHANNELS :",ch,"SLICES :",sl,"FRAMES :",fr);

setBatchMode(true);
// create maximum & minmum intensity projetion
run("Z Project...", "start=1 stop="+toString(fr)+" projection=[Max Intensity]");
getStatistics(areaMax, meanMax, minMax, maxMax);
selectWindow(title_orig);
run("Z Project...", "start=1 stop="+toString(fr)+" projection=[Min Intensity]");
// determine the maximum pixel value to adjust the y-scaling of the plot
getStatistics(areaMin, meanMin, minMin, maxMin);
close();

print("Min / Max : ",minMin," / ",maxMax);

Dialog.create("Multiple ROI Kinetics");
Dialog.addMessage("Version Number: " + toString(version_number,1));
Dialog.addNumber("Time per frame [ms]",1);
Dialog.addCheckbox("Plot Data Points", false);
Dialog.addNumber("Line Width for Lines",2);
Dialog.addNumber("Line Width for ROIs",2);
//Dialog.addCheckbox("Inculde Min & Max in result table", false);
Dialog.addCheckbox("ROIs added via Time Series Analyzer", false);
Dialog.addCheckbox("Save Results as TXT-File", false);
Dialog.addCheckbox("Save Results as XLS-File", false);
Dialog.addCheckbox("Show Results in Python - MatPlotLib", false);

Dialog.show();
timeframe = Dialog.getNumber();
plotpoints = Dialog.getCheckbox();
lw_plot = Dialog.getNumber();
lw_roi = Dialog.getNumber();
//minmax = Dialog.getCheckbox();
roiadd = Dialog.getCheckbox();
savetxt = Dialog.getCheckbox();
savexls = Dialog.getCheckbox();
openmpl = Dialog.getCheckbox();

run("Set Measurements...", "  mean display redirect=None decimal=3");
//if (minmax == true){
//	run("Set Measurements...", "  mean min display redirect=None decimal=3");
//	}
//else if (minmax == false){
//	run("Set Measurements...", "  mean display redirect=None decimal=3");	
//	}

// specify plot dimensions
run("Profile Plot Options...", "width=600 height=400 minimum=0 maximum=0 draw");

numROI = roiManager("Count");
print("NumROI :  ",numROI);
if (numROI >= 9) {
	showMessage("Error Message","Too many ROIs - only 8 are possible ...");
	exit();
	}

frames = newArray(fr);
roi1  = newArray(fr); // ROI number 1
roi2  = newArray(fr);
roi3  = newArray(fr);
roi4  = newArray(fr);
roi5  = newArray(fr);
roi6  = newArray(fr);
roi7  = newArray(fr);
roi8  = newArray(fr);

if (roiadd == true) {
	lb = newArray("Mean(ROI001)","Mean(ROI002)","Mean(ROI003)","Mean(ROI004)","Mean(ROI005)","Mean(ROI006)","Mean(ROI007)","Mean(ROI008)");	
}
if (roiadd == false) {
	lb = newArray("Mean1","Mean2","Mean3","Mean4","Mean5","Mean6","Mean7","Mean8");
}

// create color list for ROIs
List.set("1","red"); // color for ROI 1
List.set("2","green");
List.set("3","blue");
List.set("4","magenta");
List.set("5","orange");
List.set("6","cyan");
List.set("7","yellow");
List.set("8","black");

// adjust color properties for all ROIs
for (i=1; i<=numROI; i++) {
	roiManager("Select", i-1);
	colorvalue = List.get(toString(i));
	roiManager("Set Color", colorvalue);
	roiManager("Set Line Width", lw_roi);
	}

roiManager("Deselect");
roiManager("Multi Measure");

// create array containing the frame numbers
for (i=1; i<=fr; i++) {
	frames[i-1] = i * timeframe;
	//setResult("Time", i-1, i * timeframe);
	setResult("Time", i-1, frames[i-1]);
	}


for (i=1; i<=fr; i++) {
	if (numROI == 1) {
		roi1[i-1] = getResult(lb[0],i-1);
		}
	if (numROI == 2) {
		roi1[i-1] = getResult(lb[0],i-1);
		roi2[i-1] = getResult(lb[1],i-1);
		}
	if (numROI == 3) {
		roi1[i-1] = getResult(lb[0],i-1);
		roi2[i-1] = getResult(lb[1],i-1);
		roi3[i-1] = getResult(lb[2],i-1);	
		}
	if (numROI == 4) {
		roi1[i-1] = getResult(lb[0],i-1);
		roi2[i-1] = getResult(lb[1],i-1);
		roi3[i-1] = getResult(lb[2],i-1);
		roi4[i-1] = getResult(lb[3],i-1);
		}
	if (numROI == 5) {
		roi1[i-1] = getResult(lb[0],i-1);
		roi2[i-1] = getResult(lb[1],i-1);
		roi3[i-1] = getResult(lb[2],i-1);
		roi4[i-1] = getResult(lb[3],i-1);
		roi5[i-1] = getResult(lb[4],i-1);
		}
	if (numROI == 6) {
		roi1[i-1] = getResult(lb[0],i-1);
		roi2[i-1] = getResult(lb[1],i-1);
		roi3[i-1] = getResult(lb[2],i-1);
		roi4[i-1] = getResult(lb[3],i-1);
		roi5[i-1] = getResult(lb[4],i-1);
		roi6[i-1] = getResult(lb[5],i-1);
		}
	if (numROI == 7) {
		roi1[i-1] = getResult(lb[0],i-1);
		roi2[i-1] = getResult(lb[1],i-1);
		roi3[i-1] = getResult(lb[2],i-1);
		roi4[i-1] = getResult(lb[3],i-1);
		roi5[i-1] = getResult(lb[4],i-1);
		roi6[i-1] = getResult(lb[5],i-1);
		roi7[i-1] = getResult(lb[6],i-1);	
		}
	if (numROI == 8) {
		roi1[i-1] = getResult(lb[0],i-1);
		roi2[i-1] = getResult(lb[1],i-1);
		roi3[i-1] = getResult(lb[2],i-1);
		roi4[i-1] = getResult(lb[3],i-1);
		roi5[i-1] = getResult(lb[4],i-1);
		roi6[i-1] = getResult(lb[5],i-1);
		roi7[i-1] = getResult(lb[6],i-1);
		roi8[i-1] = getResult(lb[7],i-1);		
		}
	}

// do the plots
Plot.create("Line Plot Test", "Frame Number / Time [ms]", "Mean Intensity [cts]", frames, roi1);
Plot.setLimits(1, fr*timeframe*1.02, minMin*1.3, maxMax*1.1);
//Plot.setLimits(1, fr*timeframe*1.02, minMin, maxMax*0.8);
Plot.setLineWidth(lw_plot);
if (plotpoints == true) {
	Plot.add("Circle", frames, roi1);	
	}
// ROI 2
if (numROI >= 2) {
	Plot.setColor(List.get(toString(2)));
	Plot.add("Line", frames, roi2);
	if (plotpoints == true) {
		Plot.add("Circle", frames, roi2);	
		}
}
// ROI 3
if (numROI >= 3) {
	Plot.setColor(List.get(toString(3)));
	Plot.add("Line", frames, roi3);
	if (plotpoints == true) {
		Plot.add("Circle", frames, roi3);	
	}
}
// ROI 4
if (numROI >= 4) {
	Plot.setColor(List.get(toString(4)));
	Plot.add("Line", frames, roi4);
	if (plotpoints == true) {
		Plot.add("Circle", frames, roi4);	
	}
}
// ROI 5
if (numROI >= 5) {
	Plot.setColor(List.get(toString(5)));
	Plot.add("Line", frames, roi5);
	if (plotpoints == true) {
		Plot.add("Circle", frames, roi5);	
	}
}
// ROI 6
if (numROI >= 6) {
	Plot.setColor(List.get(toString(6)));
	Plot.add("Line", frames, roi6);
	if (plotpoints == true) {
		Plot.add("Circle", frames, roi6);	
	}
}
// ROI 7
if (numROI >= 7) {
	Plot.setColor(List.get(toString(7)));
	Plot.add("Line", frames, roi7);
	if (plotpoints == true) {
		Plot.add("Circle", frames, roi7);	
	}
}
// ROI 8
if (numROI == 8) {
	Plot.setColor(List.get(toString(8)));
	Plot.add("Line", frames, roi8);
	if (plotpoints == true) {
		Plot.add("Circle", frames, roi8);	
	}
}

// ROI 1
Plot.setColor(List.get(toString(1)));
// display the plot
Plot.show();
setBatchMode(false);

if (savetxt == true) {
   	if (nResults==0) {
   		exit("Results table is empty");
   	}
   	else {
   		path = imagedir + imagefilename + "_Results.txt";
   		//print(path);
   		saveAs("Measurements", path);
   	}
}

if (savexls == true) {
	// save contents of Results table in Excel
   	if (nResults==0) {
   		exit("Results table is empty");
   	}
   	else {
   		path = imagedir + imagefilename + "_Results.xls";
   		//print(path);
   		saveAs("Measurements", path);
   		//exec("cmd", "/c", "start", "excel.exe", "c:\\Results.xls");
   	}
}

if (openmpl == true) {
   	if (nResults==0) {
   		exit("Results table is empty");
   	}
   	else {
   		path = imagedir + imagefilename + "_Results.txt";
   		saveAs("Measurements", path);
   		exec("cmd", "/c","start", "python.exe", "c:/Dokumente und Einstellungen/sebastian.rhode/Eigene Dateien/Sebi/Projects_TILL/LA_Software/Online_Analysis/kinetics_FIJI.py", "run");
   	}
}