doesn't recognize ratio command?
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doesn't recognize ratio command?
 
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Hi all,
A very simple question.......I would like to use intensity correlation analysis in the MBF bundle with the latest Image J v1.38. The plugins show up in the pulldown menu on my Mac (tiger not leopard OSX), I can run all the plugins in the colocalization analysis folder but ICA and Manders. The ICA plug in comes back and says " no ratio command". Any ideas? I know this is a dumb question,but I've reloaded everything several times and it still does it. Thanks, Gretchen Unger [hidden email] |
Re: doesn't recognize ratio command?
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Ratio.lut is a lookup table included with the MBF bundle in both the
lut and luts folders. The plugins only version, however, is missing the luts folder. As a result, there is no "Ratio" command in the Image>Lookup Tables menu (LUTs in the luts folder are listed in this menu). You can work around this problem by making a copy of the lut folder and naming it luts. You can't simply change the name from lut to luts because the ICA plugin looks for another LUT (ICA.lut) in the lut folder. -wayne On Jan 13, 2008, at 8:42 PM, Gretchen Unger, Ph.D. wrote: > Hi all, > > A very simple question.......I would like to use intensity correlation > analysis in the MBF bundle with the latest Image J v1.38. The plugins > show up in the pulldown menu on my Mac (tiger not leopard OSX), I can > run all the plugins in the colocalization analysis folder but ICA and > Manders. The ICA plug in comes back and says " no ratio command". Any > ideas? I know this is a dumb question,but I've reloaded everything > several times and it still does it. > > Thanks, > Gretchen Unger > [hidden email] > |
Re: doesn't recognize ratio command?
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Hi all,
Still trying to get the ICA plugin to run. I have put a copy of Ratio.lut in a folder entitled "luts" outside the plug-in folder and inside the plug-in folder in subfolders entitled "LUT" and "luts". Of these last two folders, the LUT folder had lots of files in it while the luts folder had not so many, all of which had a dark icon different looking from the files in the "luts" folder outside the plugin folder. The plug-in comes back with the error message "unrecognized command: ratio". Thanks in advance for any thoughts. gretchen >Ratio.lut is a lookup table included with the MBF bundle in both the >lut and luts folders. The plugins only version, however, is missing >the luts folder. As a result, there is no "Ratio" command in the >Image>Lookup Tables menu (LUTs in the luts folder are listed in this >menu). You can work around this problem by making a copy of the lut >folder and naming it luts. You can't simply change the name from lut >to luts because the ICA plugin looks for another LUT (ICA.lut) in >the lut folder. > >-wayne > >On Jan 13, 2008, at 8:42 PM, Gretchen Unger, Ph.D. wrote: > >>Hi all, >> >>A very simple question.......I would like to use intensity >>correlation analysis in the MBF bundle with the latest Image J >>v1.38. The plugins show up in the pulldown menu on my Mac (tiger >>not leopard OSX), I can run all the plugins in the colocalization >>analysis folder but ICA and Manders. The ICA plug in comes back and >>says " no ratio command". Any ideas? I know this is a dumb >>question,but I've reloaded everything several times and it still >>does it. >> >>Thanks, >>Gretchen Unger >>[hidden email] -- _________________________________________________________________________ Gretchen M. Unger, Ph.D. Research-151, 3q-120 VA Medical Center One Veterans Drive Minneapolis, Minnesota 55417 email: [hidden email] Tel: 612-467-5167, 612-467-2865 FAX: 612-725-2023 __________________________________________________________________________ |
Re: doesn't recognize ratio command?
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Hi Gretchen,
i know this post is kind of old, but i have encountered a similar problem than you when using the ICA plugin "unrecognized command: ratio". did you find a way around this? thanks Cedric Asensio
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Re: doesn't recognize ratio command?
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Yes, if I recall correctly, I copied the Ratio.lut into the luts/ and
lut/ folders. Also I named it BOTH Ratio.lut AND Ratio_.lut I'm sure that all of it isn't necessary, but it worked and I didn't want to waste the time trying to figure it all out. There was another problem with the ICA plugin - some of the dialog commands weren't macro friendly, so, I got the source code and modified it to be macro friendly. I can't post the fixed plugin, so I'll just email it to you directly. John Cedric Asensio wrote: > Hi Gretchen, > > i know this post is kind of old, but i have encountered a similar problem > than you when using the ICA plugin "unrecognized command: ratio". did you > find a way around this? > > > thanks > > Cedric Asensio > > > Gretchen Unger, Ph.D. wrote: >> Hi all, >> >> Still trying to get the ICA plugin to run. I have put a copy of >> Ratio.lut in a folder entitled "luts" outside the plug-in folder and >> inside the plug-in folder in subfolders entitled "LUT" and "luts". Of >> these last two folders, the LUT folder had lots of files in it while >> the luts folder had not so many, all of which had a dark icon >> different looking from the files in the "luts" folder outside the >> plugin folder. The plug-in comes back with the error message >> "unrecognized command: ratio". Thanks in advance for any thoughts. >> gretchen >> >>> Ratio.lut is a lookup table included with the MBF bundle in both the >>> lut and luts folders. The plugins only version, however, is missing >>> the luts folder. As a result, there is no "Ratio" command in the >>> Image>Lookup Tables menu (LUTs in the luts folder are listed in this >>> menu). You can work around this problem by making a copy of the lut >>> folder and naming it luts. You can't simply change the name from lut >>> to luts because the ICA plugin looks for another LUT (ICA.lut) in >>> the lut folder. >>> >>> -wayne >>> >>> On Jan 13, 2008, at 8:42 PM, Gretchen Unger, Ph.D. wrote: >>> >>>> Hi all, >>>> >>>> A very simple question.......I would like to use intensity >>>> correlation analysis in the MBF bundle with the latest Image J >>>> v1.38. The plugins show up in the pulldown menu on my Mac (tiger >>>> not leopard OSX), I can run all the plugins in the colocalization >>>> analysis folder but ICA and Manders. The ICA plug in comes back and >>>> says " no ratio command". Any ideas? I know this is a dumb >>>> question,but I've reloaded everything several times and it still >>>> does it. >>>> >>>> Thanks, >>>> Gretchen Unger >>>> [hidden email] >> >> -- >> _________________________________________________________________________ >> Gretchen M. Unger, Ph.D. >> Research-151, 3q-120 >> VA Medical Center >> One Veterans Drive >> Minneapolis, Minnesota 55417 >> >> email: [hidden email] >> Tel: 612-467-5167, 612-467-2865 >> FAX: 612-725-2023 >> __________________________________________________________________________ >> >> > -- John K. Alexander, Ph.D. Post-Doctoral Fellow William Green Laboratory University of Chicago Dept. Neurobiology, Pharmacology, and Physiology 947 East 58th Street Abott Hall 402 Chicago, IL 60637 (off) 773-702-9386 (fax) 773-702-3774 [hidden email] |
ICA Frequency scatter plot fix
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In reply to this post by Cedric Asensio
I just started to use the Intensity Correlation Analysis and like some I
had problem if the Display Frequency scatter plot option. Thanks to John Alexander information that it is simply a lut file that is not present in the standard ImageJ installation I just changed the source code so that it uses the fire lut instead, which if I'm not mistaken is in the ImageJ core. Here is the fixed source code for those interested. Gabriel Lapointe //Version 2.0 //Sept.5.05 //T.J. Collins [hidden email] //E.Stanley [hidden email] /* Nov.21.2008 Changed the frequency scatter plot lut to allow the use of the plugin without the need to the Ration.lut file modification by [hidden email] */ import java.awt.*; import java.io.*; import ij.*; import ij.gui.*; import ij.process.*; import ij.text.*; import ij.plugin.PlugIn; import java.text.DecimalFormat; import ij.measure.*; import ij.measure.ResultsTable.*; import ij.plugin.filter.*; public class Intensity_Correlation_Analysis implements PlugIn { static boolean headingsSet; private ImageStack img1, img2; private static int index1; private static int index2; private Roi roi, roi1, roi2; private ImagePlus imp1, imp2, impmask; private ImageProcessor ip1, ip2, ipmask; private static boolean displayCounts = Prefs.get("ICA_counts.boolean",false); private boolean displayRGHist = Prefs.get("ICA_RGB.boolean",false); private boolean displayFreqHist = Prefs.get("ICA_freq.boolean",true); private boolean displayICQplots = Prefs.get("ICA_ICQplots.boolean",true); private boolean displayPositives = Prefs.get("ICA_positives.boolean",true); private boolean displayPDMs =Prefs.get("ICA_displayPDMs.boolean",false); private boolean keepROIimage= Prefs.get("ICA_keepROI.boolean",true); private boolean useROI ; private int bitDepth = 255; private int dualChannelIndex = (int)Prefs.get("ICA_channels.int",0); private boolean currentSliceOnly= Prefs.get("ICA_current.boolean",true); private boolean overlayPositives=Prefs.get("ICA_overlayPositives.boolean",false); private boolean useThreshold=Prefs.get("ICA_threshold.boolean",false); private int indexRoi= (int)Prefs.get("ICA_indexRoi.int",0); private int ch2threshmin=1; private int ch1threshmin=1; private int ch2threshmax=255; private int ch1threshmax=255; private int xLoc=0; private int yLoc=0; private int dot=3; private int currentSlice ; private int nslices; private int width, height, rwidth, rheight, xOffset, yOffset; private int colIndex1 = 0; private int colIndex2 = 1; private int [] color = new int [3]; private ImageWindow winimpROI2,winimpROI1; public void run(String arg) { if (showDialog()) correlate(img1, img2); } public boolean showDialog() { int[] wList = WindowManager.getIDList(); if (wList==null) { IJ.noImage(); return false; } String[] titles = new String[wList.length]; for (int i=0; i<wList.length; i++) { ImagePlus imp = WindowManager.getImage(wList[i]); if (imp!=null) titles[i] = imp.getTitle(); else titles[i] = ""; } if (wList.length<2) { IJ.showMessage("Image Correlator", "Requires at least two images to be open"); return false; } String[] dualChannels= { "Red : Green","Red : Blue", "Green : Blue",}; String[] chooseROI= { "None","Channel 1", "Channel 2",}; if (index1>=titles.length)index1 = 0; if (index2>=titles.length)index2 = 0; GenericDialog gd = new GenericDialog("Image Correlation Analysis"); gd.addChoice("Channel_1 ", titles, titles[0]); gd.addChoice("Channel_2 ", titles, titles[1]); gd.addChoice("Channel_Combination", dualChannels, dualChannels[dualChannelIndex]); gd.addChoice("Use_ROI", chooseROI, chooseROI[indexRoi]); gd.addCheckbox("Use_Thresholds", useThreshold); gd.addCheckbox("Keep_merged ROI", keepROIimage); gd.addCheckbox("Display_Colour Scatter plot", displayRGHist); gd.addCheckbox("Display_Frequency Scatter plot", displayFreqHist); gd.addCheckbox("Display_Intensity Counts ", displayCounts); gd.addCheckbox("Current_Slice only:", currentSliceOnly); gd.addMessage("Intensity_Correlation Analysis"); gd.addCheckbox("Display_ICA_plots", displayICQplots); gd.addNumericField("Crosshair_size_(pixels)", dot,0); gd.addCheckbox("Display_PDM_Image", displayPositives); gd.addCheckbox("Display_+ve_PDMs_only", overlayPositives); gd.addCheckbox("List_PDM_values", displayPDMs); gd.addMessage("For details of Intensity Correlation Analysis (ICA) "+"\n"+"see and cite: Li, Lau, Morris, Guo & Stanley. "+"\n"+"J Neurosci. (2004) 24:4070-81. "); gd.showDialog(); if (gd.wasCanceled()) return false; index1 = gd.getNextChoiceIndex(); index2 = gd.getNextChoiceIndex(); dualChannelIndex = gd.getNextChoiceIndex(); indexRoi = gd.getNextChoiceIndex(); useThreshold = gd.getNextBoolean(); keepROIimage =gd.getNextBoolean(); displayRGHist = gd.getNextBoolean(); displayFreqHist = gd.getNextBoolean(); displayCounts = gd.getNextBoolean(); currentSliceOnly = gd.getNextBoolean(); displayICQplots =gd.getNextBoolean(); dot = (int)gd.getNextNumber(); displayPositives =gd.getNextBoolean(); overlayPositives =gd.getNextBoolean(); displayPDMs =gd.getNextBoolean(); String title1 = titles[index1]; String title2 = titles[index2]; imp1 = WindowManager.getImage(wList[index1]); imp2 = WindowManager.getImage(wList[index2]); ImageStack img1 = imp1.getStack(); ImageStack img2 = imp2.getStack(); width = img1.getWidth(); height = img1.getHeight(); if (imp1.getType()==imp1.COLOR_RGB || imp2.getType()==imp2.COLOR_RGB) { IJ.showMessage("Image Correlator", "Both images must be 8- or 16-bit grayscale."); return false; } currentSlice = imp1.getCurrentSlice(); ImageWindow winimp1= imp1.getWindow(); ImageWindow winimp2= imp2.getWindow(); ImageProcessor ipthresh1 = imp1.getProcessor(); ImageProcessor ipthresh2 = imp2.getProcessor(); if (dualChannelIndex==1) colIndex2 = 2; if (dualChannelIndex==2) {colIndex1 = 1;colIndex2 =2 ;} if (imp1.getType()!=imp1.GRAY8) {bitDepth= 65208; ch2threshmax=65208; ch1threshmax=65208; } if((useThreshold)) { if (ipthresh2.getMinThreshold()!=ipthresh2.NO_THRESHOLD) { ch2threshmin=(int)ipthresh2.getMinThreshold(); ch2threshmax=(int)ipthresh2.getMaxThreshold(); } if (ipthresh1.getMinThreshold()!=ipthresh1.NO_THRESHOLD) { ch1threshmin=(int)ipthresh1.getMinThreshold(); ch1threshmax=(int)ipthresh1.getMaxThreshold(); } } //IJ.showMessage("ch1 "+ch1threshmin+" "+ch1threshmax+"; ch2 " +ch2threshmin+" "+ ch2threshmax); nslices = imp1.getStackSize(); if (indexRoi==0) useROI = false; else useROI=true; ip1 = imp1.getProcessor(); ip2 = imp2.getProcessor(); roi1 = imp1.getRoi(); roi2 = imp2.getRoi(); Rectangle rect =ip1.getRoi(); if ((indexRoi==1)) {if(roi1==null) {useROI=false;} else { ipmask = imp1.getMask(); rect = ip1.getRoi(); } } if ((indexRoi==2)) {if(roi2==null) {useROI=false;} else {ipmask = imp2.getMask(); rect = ip2.getRoi(); } } if (useROI==false) { xOffset = 0;yOffset = 0; rwidth=width; rheight =height; } else { xOffset = rect.x; yOffset = rect.y; rwidth=rect.width; rheight =rect.height; } //.showMessage(""+useROI); int g1=0;int g2=0; return true; } public void correlate(ImageStack img1, ImageStack img2) { boolean resultsOpen = IJ.isResultsWindow(); if (!resultsOpen) headingsSet = false; String Ch1fileName = imp1.getTitle(); String Ch2fileName = imp2.getTitle(); String fileName = Ch1fileName + " and " + Ch2fileName; img1 = imp1.getStack(); img2 = imp2.getStack(); int width2 = img2.getWidth(); int height2 = img2.getHeight(); if ((height!=height2)||(width!=width2)) { IJ.showMessage("Image Correlator", "Images selected are not the same size."); return ; } String path = Menus.getPlugInsPath(); ImageProcessor ip1 = imp1.getProcessor(); ImageProcessor ip2 = imp2.getProcessor(); int cdash=0; ImageProcessor plot = new FloatProcessor(316, 296); ImageProcessor plot16bit = new ShortProcessor(316, 296); ImageProcessor plotICQch1 = new ShortProcessor(316, 296); ImageProcessor plotICQch2 = new ShortProcessor(316, 296); ImageProcessor plotICQch12 = new ShortProcessor(316, 296); ImageProcessor plotICQch22 = new ShortProcessor(316, 296); ImageProcessor ipMerge= new ColorProcessor(rwidth, rheight); ImageStack mergeStack = new ImageStack (rwidth,rheight); ImageStack plotStackPDM = new ImageStack (rwidth+35,rheight); ImageStack plotStackPDMpositives= new ImageStack (rwidth,rheight+15); ImageStack plotStackPDMnegatives= new ImageStack (rwidth,rheight+15); StringBuffer sb2 = new StringBuffer(); StringBuffer sb3 = new StringBuffer(); int ch1, z2,ch2; int sumCh1 = 0; int sumCh2 = 0; double sumX = 0; double sumXY = 0; double sumXX = 0; double sumYY = 0; double sumY = 0; double sumXtotal = 0; double sumYtotal = 0; double colocX = 0; double colocY = 0; double countX = 0; double countY = 0; int N = 0; int countnegative = 0; int countpositive = 0; int signcountch1=0; int signcountch2=0; int countZeroPair=0; int countNonZeroPair=0; int ch1max = 0; int ch2max = 0; int ch1min = 65208; int ch2min = 65208; double ch1Norm = 1; double ch2Norm = 1; double ch1normdble =0; double ch2normdble =0; DecimalFormat df4 = new DecimalFormat("##0.0000"); DecimalFormat df3 = new DecimalFormat("##0.000"); DecimalFormat df2 = new DecimalFormat("##0.00"); DecimalFormat df1 = new DecimalFormat("##0.0"); DecimalFormat df0 = new DecimalFormat("##0"); double ch2NormMax = 0; double ch1NormMax=0; double ch2NormMin = 0; double ch1NormMin=0; int PDM2plot=0; double PDMmax = 0; double PDMmin = 0; double PDM=0; double PDMrange=0; double PDMnorm=0; int count=0; int countTotal=0; int ch22Plot=128; int ch12Plot=128; int numberCh1Pixels = 0; int numberCh2Pixels=0; int numberBothNonZero=0; int mask=1; int countP=0; int scaledZ1=0; int scaledZ2=0; //run1 int countAll=0; int countZeroZero=0; // getting mean ch1 //getting mean ch2 // //IJ.showMessage("ch1 "+ch1threshmin+" "+ch1threshmax+"; ch2 " +ch2threshmin+" "+ ch2threshmax); boolean include = false; for (int i=1; i<=nslices; i++) { if(currentSliceOnly) { i = currentSlice; nslices = currentSlice; } IJ.showStatus("Correlating (1/3 steps): "+ i +"/" + nslices); ip1 = img1.getProcessor(i); ip2 = img2.getProcessor(i); for (int y=0; y<rheight; y++) { for (int x=0; x<rwidth; x++) {mask=1; if((useROI)&&(ipmask!=null)) mask = (int)ipmask.getPixelValue(x,y); if (mask!=0) { countTotal++; ch1 = (int)ip1.getPixel(x+xOffset,y+yOffset); ch2 = (int)ip2.getPixel(x+xOffset,y+yOffset); if (ch1+ch2==0) countZeroZero++; countAll++; if ((ch1>=ch1threshmin)&&(ch2>=ch2threshmin)) include = true; else include = false; if(!useThreshold) include = true; if((ch1>=ch1threshmin)) {sumCh1 = sumCh1 + ch1; if(ch1>=ch1max) ch1max = ch1; if(ch1<=ch1min) ch1min = ch1; numberCh1Pixels++; } if((ch2>=ch2threshmin)) {if(ch2>=ch2max) ch2max = ch2; if(ch2<=ch2min) ch2min = ch2; numberCh2Pixels++; sumCh2 = sumCh2 + ch2; } if(include) { sumX = sumX+ch1; sumXY = sumXY + (ch1 * ch2); sumXX = sumXX + (ch1 * ch1); sumYY = sumYY + (ch2 *ch2); sumY = sumY + ch2; countP++; } } } } } countNonZeroPair = countAll-countZeroZero; double meanCh1= (double)sumCh1/numberCh1Pixels; double meanCh2 = (double)sumCh2/numberCh2Pixels; ch1min-=1; ch1max+=1; ch2min-=1; ch2max+=1; //IJ.showMessage("ch1max: "+ch1max+" ch1min "+ch1min); // IJ.showMessage("Ch1: "+meanCh1+" Ch2: "+meanCh2+" Countnon00:"+ countNonZeroPair); double normMeanCh1 = ((double)meanCh1-(double)ch1min)/((double)ch1max-(double)ch1min); double normMeanCh2=((double)meanCh2-(double)ch2min)/((double)ch2max-(double)ch2min); int ch2Mean2Plot = (int)((((double)meanCh2-(double)ch2min)/((double)ch2max-(double)ch2min))*(double)256); int ch1Mean2Plot = (int)((((double)meanCh1-(double)ch1min)/((double)ch1max-(double)ch1min))*(double)256); //IJ.showMessage("mean ch1= "+meanCh1+"ch1 min= "+ch1min+" ch1max= "+ch1max ); if(!useThreshold) countP=countAll-countZeroZero; double pearsons1 = sumXY - (sumX*sumY/countP); double pearsons2 = sumXX - (sumX*sumX/countP); double pearsons3 = sumYY - (sumY*sumY/countP); //run2 to get PDM extreme for (int i=1; i<=nslices; i++) { IJ.showStatus("Correlating (2/3 steps): "+ i +"/" + nslices); if(currentSliceOnly) { i = currentSlice; nslices = currentSlice; } ip1 = img1.getProcessor(i); ip2 = img2.getProcessor(i); for (int y=0; y<rheight; y++) { for (int x=0; x<rwidth; x++) {mask=1; if((useROI)&&(ipmask!=null)) mask = ipmask.getPixel(x,y); if (mask!=0) { ch1 = (int)ip1.getPixel(x+xOffset,y+yOffset); ch2 = (int)ip2.getPixel(x+xOffset,y+yOffset); if ((ch1>=ch1threshmin)&&(ch2>=ch1threshmin)) include = true; else include = false; if(!useThreshold) include = true; if(include) { ch1Norm = (((double)ch1-(double)ch1min)/((double)ch1max-(double)ch1min)); ch2Norm = (((double)ch2-(double)ch2min)/((double)ch2max-(double)ch2min)); PDM = ((double)ch1Norm-(double)normMeanCh1)*((double)ch2Norm-(double)normMeanCh2); if (PDM>=PDMmax) PDMmax=PDM; if (PDM<=PDMmin) PDMmin=PDM; } } } } } if (PDMmax+PDMmin>0) PDMrange = 1.05*(PDMmax); if (PDMmax+PDMmin<0) PDMrange = -1.05*(PDMmin); //IJ.showMessage(""+PDMrange); ImageProcessor ipPDMpositives = new FloatProcessor(width,height+15); ImageProcessor ipPDMnegatives = new FloatProcessor(width,height+15); //run 3 to clculate final coefficients and plots countP=0; sumX = 0; sumXY = 0; sumXX = 0; sumYY = 0; sumY = 0; PDM=0; int countInclude=0; countZeroPair=0; countpositive=0; for (int i=1; i<=nslices; i++) { ImageProcessor ipPDM = new FloatProcessor(rwidth+35,rheight); ipPDMpositives = new FloatProcessor(rwidth,rheight+15); ipPDMnegatives = new FloatProcessor(rwidth,rheight+15); if(currentSliceOnly) { i = currentSlice; nslices = currentSlice; } IJ.showStatus("Correlating (3/3 steps): "+ i +"/" + nslices); ip1 = img1.getProcessor(i); ip2 = img2.getProcessor(i); for (int y=0; y<=rheight; y++) { for (int x=0; x<=rwidth; x++) {mask=1; if((useROI)&&(ipmask!=null)) mask = ipmask.getPixel(x,y); if (mask!=0) { ch1 = (int)ip1.getPixel(x+xOffset,y+yOffset); ch2 = (int)ip2.getPixel(x+xOffset,y+yOffset); if (ch1+ch2==0) countZeroPair++; //crreate merged image color[colIndex1]=(int)((ch1*bitDepth)/bitDepth); color[colIndex2]=(int)((ch2*bitDepth)/bitDepth); ipMerge.putPixel(x,y,color); sumXtotal = sumXtotal+ch1; sumYtotal = sumYtotal+ch2; if((ch2>=ch2threshmin)&(ch2<=ch2threshmax)) { colocX = colocX + ch1; countY = countY + 1; } if((ch1threshmin<=ch1)&(ch1<=ch1threshmax)) { colocY = colocY + ch2; countX = countX + 1; } if ((ch1threshmin<=ch1)&&(ch1<=ch1threshmax)) numberCh1Pixels++; if ((ch2threshmin<=ch2)&&(ch2<=ch2threshmax)) numberCh2Pixels++; if(((ch1>=ch1threshmin)&&(ch2>=ch2threshmin))||!useThreshold) { countInclude++; ipPDM.putPixel(x+35,y,0);countP++; sumX = sumX+ch1; sumXY = sumXY + (ch1 * ch2); sumXX = sumXX + (ch1 * ch1); sumYY = sumYY + (ch2 * ch2); sumY = sumY + ch2; ch1Norm = (((double)ch1-ch1min)/(ch1max-ch1min)); ch2Norm = (((double)ch2-ch2min)/(ch2max-ch2min)); PDMnorm =((double)ch1Norm-(double)normMeanCh1)*((double)ch2Norm-(double)normMeanCh2); PDM = ((double)ch1-(double)meanCh1)*((double)ch2-(double)meanCh2); if ((PDM >0)&&(ch1+ch2!=0)) countpositive++; //PDM2plot = 127+(int)((((double)PDMnorm/(double)PDMrange))*127); PDM2plot = (int)(128+(128*(PDMnorm/PDMrange))); ch12Plot = (int)((((double)ch1-((double)ch1min))/(((double)ch1max)-((double)ch1min)))*(double)256); ch22Plot = (int)((((double)ch2-((double)ch2min))/(((double)ch2max)-((double)ch2min)))*(double)256); sb2.append(x+"\t"+y+"\t"+df4.format(PDMnorm )+"\t"+ch1+"\t"+ch2+"\n"); ipPDM.putPixelValue(x+35,y,PDMnorm); if(PDM>0) {//ipPDMpositives.putPixelValue(x,y,PDMnorm); if ((ch1>meanCh1)&&(ch2>meanCh2)) ipPDMpositives.putPixelValue(x,y,PDMnorm); if ((ch1<meanCh1)&&(ch2<meanCh2)) ipPDMnegatives.putPixelValue(x,y,PDMnorm); } signcountch1 = plotICQch12.getPixel(PDM2plot+45, 275-ch12Plot ); signcountch1++; signcountch2 = plotICQch22.getPixel(PDM2plot +45,275-ch22Plot ); signcountch2++; plotICQch1.putPixel(PDM2plot+45,275-ch12Plot ,signcountch1); plotICQch2.putPixel(PDM2plot+45,275-ch22Plot ,signcountch2); int dotWidth = (dot-1)/2; //graph plots plotICQch12.putPixel(PDM2plot+45,275-ch12Plot ,signcountch1); plotICQch22.putPixel(PDM2plot+45,275-ch22Plot ,signcountch2); for (int q=0;q<dot;q++) { plotICQch1.putPixel(PDM2plot+45-dotWidth+q,275-ch12Plot ,signcountch1); plotICQch2.putPixel(PDM2plot+45-dotWidth+q,275-ch22Plot ,signcountch2); plotICQch1.putPixel(PDM2plot+45,275-dotWidth+q-ch12Plot ,signcountch1); plotICQch2.putPixel(PDM2plot+45,275-dotWidth+q-ch22Plot ,signcountch2); } scaledZ1= (int)((double)256*(((double)ch1-(double)ch1min)/((double)ch1max-(double)ch1min))); scaledZ2= 275-(int)((double)256*(((double)ch2-(double)ch2min)/((double)ch2max-(double)ch2min))); count = plot.getPixel(scaledZ1+45, scaledZ2); count++; plot.putPixel(scaledZ1+45, scaledZ2, count); if (count<65535) count++; plot16bit.putPixel(scaledZ1+45, scaledZ2, count); } } } } //add ramp double scale = (rheight/2)/ (2*PDMrange); for (int y=1; y<(double)(rheight/2); y++) {for (int x=1; x<25; x++) { ipPDM.putPixelValue(5+x, (int)(rheight/4)+y, (double)(PDMrange-(y/scale))); } } //add text int fontSize = 12; int fontDisplacement =(int)((12*0.934211)+1.10526+4); ipPDM.drawString("+"+df2.format(PDMrange),1,(int)(rheight/4)); ipPDM.drawString(df2.format(-PDMrange),1, (int)(0.75*rheight)+fontDisplacement); ipPDMpositives.setColor((int)PDMrange); ipPDMnegatives.setColor((int)PDMrange); ipPDM.setMinAndMax(-PDMrange, PDMrange); ipPDMpositives.setMinAndMax(0, PDMrange/2); ipPDMnegatives.setMinAndMax(0, PDMrange/2); ipPDMpositives.drawString("+ve � +ve",10,(int)rheight-fontDisplacement+30); ipPDMnegatives.drawString("-ve � -ve",10,(int)rheight-fontDisplacement+30); plotStackPDM.addSlice("PDM Stack", ipPDM); plotStackPDMpositives.addSlice("+vePDMs (+ � +)", ipPDMpositives); plotStackPDMnegatives.addSlice("+vePDM (- � -)", ipPDMnegatives); mergeStack.addSlice("Merged ROI", ipMerge); } //collate positives for (int i=1; i<=nslices;i++) {if(currentSliceOnly) { nslices = 1; } ip1 = plotStackPDMnegatives.getProcessor(i); ip1.setMinAndMax(0, PDMrange); plotStackPDMpositives.addSlice("+vePDM (- � -)", ip1); } double r = pearsons1/(Math.sqrt(pearsons2*pearsons3)); plot.invertLut(); plot.resetMinAndMax(); plot16bit.resetMinAndMax(); String ch1String = " ch1"; String ch2String = " ch2"; if (dualChannelIndex!=0) ch2String = " blue"; if (dualChannelIndex==2) ch1String = " ch2"; Font font; font = new Font("SansSerif", Font.PLAIN, 12); int labelx=169; int labelx2=30; int labelx3=290; int labely=295; String labelCh1Max=df0.format(ch1max); String labelCh2Max=df0.format(ch2max); String ch1MeanLabel = df1.format(meanCh1); String ch2MeanLabel = df1.format(meanCh2); String ch2MinLabel = df0.format(ch2min); String ch1MinLabel = df0.format(ch1min); int ch1AxisOffset = ip1.getStringWidth(labelCh1Max); int ch2AxisOffset = ip1.getStringWidth(labelCh2Max); int ch1AxisOffset2 = ip1.getStringWidth(ch1MinLabel ); int ch2AxisOffset2 = ip1.getStringWidth(ch2MinLabel ); int ch1AxisOffset3 = ip1.getStringWidth(ch1MeanLabel ); int ch2AxisOffset3 = ip1.getStringWidth(ch2MeanLabel ); int w=10; String s="0"; String s1= "-"+df2.format(PDMrange); String s2 = df2.format(PDMrange); String s3= "-"+df2.format(PDMrange); String s4 = df2.format(PDMrange); if (displayFreqHist) { int plotaxis = (int) plot16bit.getMax(); plot16bit.setColor(plotaxis); plot16bit.drawString(ch1String ,150,295); plot16bit.drawString(ch2String ,1,150); plot16bit.drawString(ch1MinLabel ,39-ch1AxisOffset2+w,295); plot16bit.drawString(ch2MinLabel ,labelx2-ch2AxisOffset2+w,labely-12); plot16bit.drawString(labelCh1Max,300-ch1AxisOffset+w,295); plot16bit.drawString(labelCh2Max,labelx2-ch2AxisOffset+w,labely-268); for (int c=20; c<280; c++) { plot16bit.putPixel(45, c,plotaxis ); plot16bit.putPixel(300, c,plotaxis ); } for (int c=42; c<300; c++) { plot16bit.putPixel(c,20,plotaxis ); plot16bit.putPixel(c,275,plotaxis ); } new ImagePlus("Correlation Plot", plot16bit).show(); ImagePlus imp3 = WindowManager.getCurrentImage(); ImageWindow winimp3 = imp3.getWindow(); imp3.setTitle(fileName + " Freq. CP"); WindowManager.setCurrentWindow(winimp3); IJ.run("Enhance Contrast", "saturated=0.2 normalize"); IJ.run("Fire"); } if (displayRGHist) { ColorProcessor ipCol = new ColorProcessor(316, 296); ipCol.setColor(0xffffff); for (int y=0; y<height; y++) { for (int x=0; x<width; x++) { ch1 = (int)plot16bit.getPixel(x+45,275-y); if(ch1>0) {color[colIndex1]=x; color[colIndex2]=y; ipCol.putPixel(x+45,275-y,color); } } } for (int c=0; c<=2;c++) { color [c]= 255; } ipCol.drawString(ch1String ,165,295); ipCol.drawString(ch2String ,10,150); ipCol.drawString(ch1MinLabel ,39-ch1AxisOffset2+w,295); ipCol.drawString(ch2MinLabel ,labelx2-ch2AxisOffset2+w,labely-12); ipCol.drawString(labelCh1Max,300-ch1AxisOffset+w,295); ipCol.drawString(labelCh2Max,labelx2-ch2AxisOffset+w,labely-268); for (int c=20; c<280; c++) { ipCol.putPixel(45, c,color ); ipCol.putPixel(300, c,color ); } for (int c=42; c<300; c++) { ipCol.putPixel(c,20,color ); ipCol.putPixel(c,275,color ); } new ImagePlus("Correlation Plot", ipCol).show(); ImagePlus imp4 = WindowManager.getCurrentImage(); ImageWindow winimp4= imp4.getWindow(); IJ.run("Enhance Contrast", "saturated=0.1 normalize"); imp4.setTitle(fileName + " Freq. CP"); } if (displayICQplots){ plotICQch1.resetMinAndMax(); plotICQch2.resetMinAndMax(); int ch1axis = (int) plotICQch1.getMax(); int ch2axis = (int) plotICQch2.getMax(); for (int c=20; c<280; c++) { plotICQch1.putPixel(45, c,ch1axis); plotICQch2.putPixel(45,c,ch2axis ); plotICQch1.putPixel(173, c,ch1axis); plotICQch2.putPixel(173,c,ch2axis ); plotICQch1.putPixel(300, c,ch1axis); plotICQch2.putPixel(300,c,ch2axis ); } for (int c=42; c<300; c++) { plotICQch1.putPixel(c,20,ch1axis); plotICQch2.putPixel(c,20,ch2axis ); plotICQch1.putPixel(c,275,ch1axis); plotICQch2.putPixel(c,275,ch2axis ); cdash++; if (cdash==2) { plotICQch1.putPixel(c,275-ch1Mean2Plot,ch1axis); plotICQch2.putPixel(c,275-ch2Mean2Plot,ch2axis); cdash=0; } } plotICQch2.setFont(font); plotICQch1.setFont(font); plotICQch2.setColor(ch2axis); plotICQch2.drawString(s ,labelx+1,labely); plotICQch2.drawString(s1,labelx2,labely); plotICQch2.drawString(s2,labelx3,labely); plotICQch1.setColor(ch1axis); plotICQch1.drawString(s,labelx+1,labely); plotICQch1.drawString(s3,labelx2,labely); plotICQch1.drawString(s4,labelx3,labely); //add titles plotICQch1.drawString(Ch1fileName ,labelx2-ch1AxisOffset2+w+20,18); plotICQch2.drawString(Ch2fileName ,labelx2-ch1AxisOffset2+w+20,18); //add y axis labels plotICQch1.drawString(ch1MinLabel ,labelx2-ch1AxisOffset2+w,labely-12); plotICQch2.drawString(ch2MinLabel ,labelx2-ch2AxisOffset2+w,labely-12); plotICQch1.drawString(labelCh1Max,labelx2-ch1AxisOffset+w,labely-268); plotICQch2.drawString(labelCh2Max,labelx2-ch2AxisOffset+w,labely-268); plotICQch2.drawString(ch2MeanLabel,labelx2-ch2AxisOffset3+w,282-ch2Mean2Plot); plotICQch1.drawString(ch1MeanLabel,labelx2-ch1AxisOffset3+w,282-ch1Mean2Plot); new ImagePlus("ICA plot -"+Ch1fileName +": "+ ch1String , plotICQch1).show(); IJ.run("Enhance Contrast", "saturated=0.0 equalize"); ImagePlus ICQch1 = WindowManager.getCurrentImage(); new ImagePlus("ICA plot -"+Ch2fileName +": "+ ch2String , plotICQch2).show(); IJ.run("Enhance Contrast", "saturated=0.0 equalize"); ImagePlus ICQch2 = WindowManager.getCurrentImage(); ImageWindow winICQch1 = ICQch1.getWindow(); ImageWindow winICQch2 = ICQch2.getWindow(); WindowManager.setCurrentWindow(winICQch2); IJ.run("Cyan"); if (dualChannelIndex==0) IJ.run("Green"); WindowManager.setCurrentWindow(winICQch1); IJ.run("Red"); if (dualChannelIndex==2) IJ.run("Green"); } if (displayCounts) displayCounts(plot); if(overlayPositives ) {new ImagePlus("+vePDM Values - " +fileName , plotStackPDMpositives).show(); IJ.run("Fire"); } if (keepROIimage)new ImagePlus("Merged ROI - "+fileName , mergeStack).show(); if (displayPositives) { String lutDir = System.getProperty("user.dir")+File.separator+"lut"+File.separator; new ImagePlus("PDM Values - "+fileName , plotStackPDM).show(); IJ.run("LUT... ", "open="+"'"+lutDir+"ICA.lut"+"'"); } if(displayPDMs) new TextWindow( "PDMs", "X-loc\tY-loc\tPDM\tCh1\tCh2", sb2.toString(),300, 400); double overlap = sumXY / (Math.sqrt(sumXX*sumYY)); double k1 = sumXY/sumXX; double k2 = sumXY/sumYY; double colocM1 = colocX/sumXtotal; double colocM2 = colocY/sumYtotal; double pixelratio = (double)countX/(double)countY; //calculate ICQ if (!useThreshold) countInclude -=countZeroPair; //IJ.showMessage("count+ = "+countpositive +" CountNonZeroPair= "+countNonZeroPair); double ICQ = ((double)countpositive/(double)countInclude)-0.5; countnegative = countNonZeroPair-countpositive; //if (countZeroPair==0) IJ.showMessage("Did you background-correct both images? \nThere appears to be no zero-zero pixel pairs"); //calculate "percentage overlap" //int percCh1 = 100*numberBothNonZero/(numberCh1Pixels); //int percCh2 = 100*numberBothNonZero/(numberCh2Pixels); String useROIstring = ""; if (useROI) useROIstring = "\nROI used: x "+xLoc+" y"+yLoc+" w"+width+" h"+height; StringBuffer ResultsOutput = new StringBuffer(); String Headings = "Image\tRr\tR\tch1:ch2\tM1\tM2\tN+ve\tNtotal\tICQ\tCh1 Thresh\tCh2 Thresh\n"; if ((!headingsSet)){ IJ.setColumnHeadings(Headings); headingsSet = true; IJ.write(Headings); } useROIstring =""; if (currentSliceOnly) useROIstring = " z"+currentSlice; if(!useThreshold) {ch1threshmin=0; ch1threshmax=0;ch2threshmin=0;ch2threshmax=0;} IJ.write(fileName+" x "+xLoc+" y"+yLoc+ useROIstring+" w"+width+" h"+height+"\t"+df3.format(r) +"\t" +df3.format(overlap)+ "\t" + df3.format(pixelratio) + "\t" +df3.format(colocM1)+ "\t" +df3.format(colocM2)+ "\t" +df0.format(countpositive)+ "\t" +df0.format(countNonZeroPair)+ "\t" + df3.format(ICQ)+ "\t" + df0.format(ch1threshmin)+"; "+df0.format(ch1threshmax)+ "\t" + df0.format(ch2threshmin)+"; "+df0.format(ch2threshmax) ); Prefs.set("ICA_ROI.boolean", useROI); Prefs.set("ICA_RGB.boolean",displayRGHist ); Prefs.set("ICA_freq.boolean",displayFreqHist ); Prefs.set("ICA_ICQplots.boolean",displayICQplots ); Prefs.set("ICA_positives.boolean",displayPositives ); Prefs.set("ICA_PDMs.boolean",false); Prefs.set("ICA_channels.int", (int)dualChannelIndex ); Prefs.set("ICA_current.boolean",currentSliceOnly); Prefs.set("ICA_overlayPositives.boolean",overlayPositives); Prefs.set("ICA_counts.boolean",displayCounts); Prefs.set("ICA_displayPDMs.boolean",displayPDMs); Prefs.set("ICA_threshold.boolean", useThreshold); Prefs.set("ICA_keepROI.boolean", keepROIimage); Prefs.set("ICA_indexRoi.int",indexRoi); imp1.changes = false; imp2.changes = false; IJ.setBackgroundColor( 0, 0,0); IJ.setForegroundColor(255,255,255 ); //imp1.setRoi(roi); IJ.showStatus("Done"); //new ImagePlus("ICA plot -"+Ch2fileName +": "+ ch2String , plotICQch22).show(); System.gc(); } void displayCounts(ImageProcessor plot) { StringBuffer sb = new StringBuffer(); int count; for (int x=0; x<256; x++) for (int y=255; y>=0; y--) { count = plot.getPixel(x,y); if (count>0) sb.append(x+"\t"+(255-y)+"\t"+count+"\n"); } new TextWindow( "Pixel intensities and frequencies", "Ch1\tCh2\tFreq.", sb.toString(), 300, 400); } } |
Biased/stretched LUTs in ImageJ
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Hi,
I have a calculated image formed by the ratiometic combination of two other images. I want to preserve the resulting pixel intensities in the calculated image which is 32-bit. I have several calculated images like these and their pixel intensities vary widely. I'd like to display these images with a colored look up table but be able to stretch LUT to bias the contrast to certain values for each image in a similar manner to what was done in Figure 3 and Figure 5 of the following paper: Parasassi, T., E. Gratton, W.M. Yu, P. Wilson, and M. Levi, 1997. Two- photon fluorescence microscopy of Laurdan generalized polarization domains in model and natural membranes. Biophysical Journal 72: 2413-2429. When I take my calculated images and add a colored calibration bar for the LUT, I can adjust the brightness and contrast to get the desired effect, but the pixel intensities no longer match the original calculated image. Is there a way to do this properly in ImageJ without altering my pixel intensities? John Oreopoulos |
Re: Biased/stretched LUTs in ImageJ
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John,
I'm not entirely sure what you mean, but Figures 5 and 3 of that paper do not contain any pixel intensity information. From my knowledge of Enrico's program, these Images were first threshholded and then false colored according to their GP values with threshholded values showing up as black. You could make something like this in ImageJ by first masking your GP image with a threshholded intensity image the then false coloring the resulting image with a LUT that shows low intensity values as black. Unfortunately, if you have 0.0 GP values, they will also show up as black. Alternatively, you could create the intensity threshhold mask, then create the false color RGB image, split it into R, G, and B images using RGB split, then mask each of these images and finally recombine them using RGB merge. There may be an RGB mask plugin out there that would do this without the split/merge steps. Jay Unruh Stowers Institute for Medical Research -----Original Message----- From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of John Oreopoulos Sent: Friday, November 21, 2008 11:54 AM To: [hidden email] Subject: Biased/stretched LUTs in ImageJ Hi, I have a calculated image formed by the ratiometic combination of two other images. I want to preserve the resulting pixel intensities in the calculated image which is 32-bit. I have several calculated images like these and their pixel intensities vary widely. I'd like to display these images with a colored look up table but be able to stretch LUT to bias the contrast to certain values for each image in a similar manner to what was done in Figure 3 and Figure 5 of the following paper: Parasassi, T., E. Gratton, W.M. Yu, P. Wilson, and M. Levi, 1997. Two- photon fluorescence microscopy of Laurdan generalized polarization domains in model and natural membranes. Biophysical Journal 72: 2413-2429. When I take my calculated images and add a colored calibration bar for the LUT, I can adjust the brightness and contrast to get the desired effect, but the pixel intensities no longer match the original calculated image. Is there a way to do this properly in ImageJ without altering my pixel intensities? John Oreopoulos |
Re: Biased/stretched LUTs in ImageJ
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In reply to this post by John Oreopoulos
John
You may find window and level (W&L) useful. If you apply a LUT to a 32-bit image, the LUT is 'stretched' to the current W&L. So, if you have a gradient of 32-bit values somewhere in your image, the LUT is applied to it - and the image pixels - 'automatically'. You can make a gradient by going File->New->Image... and selecting 32-bit and ramp. Mike John Oreopoulos wrote: > Hi, > > I have a calculated image formed by the ratiometic combination of two > other images. I want to preserve the resulting pixel intensities in the > calculated image > which is 32-bit. I have several calculated images like these and their > pixel intensities vary widely. I'd like to display these images with a > colored look up table > but be able to stretch LUT to bias the contrast to certain values for > each image in a similar manner to what was done in Figure 3 and Figure 5 > of the > following paper: > > Parasassi, T., E. Gratton, W.M. Yu, P. Wilson, and M. Levi, 1997. > Two-photon fluorescence microscopy of Laurdan generalized polarization > domains in model and natural membranes. Biophysical Journal 72: 2413-2429. > > When I take my calculated images and add a colored calibration bar for > the LUT, I can adjust the brightness and contrast to get the desired > effect, but the > pixel intensities no longer match the original calculated image. Is > there a way to do this properly in ImageJ without altering my pixel > intensities? > > John Oreopoulos |
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