Sirius Red staining Macro

[Gonzalo Bouchet]

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The author has deleted this message.

[Barry DeZonia]

Hi Gonzalo,

Are you making sure that the file you open is an RGB image (rather than an
8 or 16 or 32 bit gray image)?

On Tue, Jul 31, 2012 at 7:23 PM, Gonzalo Bouchet
<[hidden email]>wrote:

> Hello! I've downloaded a macro to quantify the are occupied by Sirius Red
> staining. When I applied, an error appears: '''Coneverts stack to images'
> requieres a stack". I'm just begging to use the ImageJ so I don't
> understand
> many things!
>
> Thanks for you help!
>
> The macro is this:
>
> // Macro for calculating percentage of fibrose componnent compared to
> cardiomyocyte component
> // taking into account the lumen part or not.
> //
> // The colour thresholding algorithm used by this macro is based on an
> algorithm written by G. Landini (version v1.8) available at:
> // http://www.dentistry.bham.ac.uk/landinig/software/software.html
>
> //
> // Note: This only works with Black background and White foreground!
> run("Colors...", "foreground=white background=black selection=yellow");
> run("Options...", "iterations=1 black count=1");
>
> //
> // All image calculation using logical operators take zero values as zero
> and 255 as "one" in the case below
> // So inverted luts only change black to white and white to black but zero
> stays zero meaning black was zero and after
> // inverting the LUT white is now zero.
> // Anything measured needs to have value 255
> // So 255 or '1' is the object
>
> // Creation 17-07-2007 J.A.M. Belien
> // Update history:
> // Small modifications with respect to version updates:
> // - last modification: modified to operate correctly with latest version
> 1.43u, dd 19-07-2010
>
> var  ExclusionImage, OriginalImage, LumenImage,
> FibrosisImage,CardioMyocyteImage;
> var exclude_high_th,lumen_low_th,fibrosis_high_th;
> var totarea_lumen,totarea_fibrosis,totarea_cardio,totarea_exclusion;
> var image_name;
>
> macro 'Assess lumen, fibrosis and cardiomyocyte components' {
>
>         // Required ImageJ version
>         requires("1.43u");
>
>         // Initialisation based on emperical analyses of test data set
>         exclude_high_th=72;
>         lumen_low_th=240;
>         fibrosis_high_th=130;
>
>         // Call open() to let user choose which image to open and analyse
>         open();
>
>         image_name = File.name;
>         OriginalImage = getImageID();
>
>         // Create duplicate and determine if green marked areas can be
> detected as
> areas to be excluded
>         run("Duplicate...", "title=exclude_area");
>
>         // Obtain (possible) exclusion area
>         ExclusionImage = getImageID();
>         selectImage(ExclusionImage);
>         min=newArray(3);
>         max=newArray(3);
>         filter=newArray(3);
>         a=getTitle();
>         run("RGBtoLab ");
>         run("RGB Stack");
>         run("Convert Stack to Images");
>         selectWindow("Red");
>         rename("0");
>         selectWindow("Green");
>         rename("1");
>         selectWindow("Blue");
>         rename("2");
>         min[0]=0;
>         max[0]=255;
>         filter[0]="pass";
>         min[1]=0;
>         max[1]=exclude_high_th;
>         filter[1]="pass";
>         min[2]=0;
>         max[2]=255;
>         filter[2]="pass";
>         for (i=0;i<3;i++){
>           selectWindow(""+i);
>           setThreshold(min[i], max[i]);
>           run("Make Binary", "thresholded remaining");
>           if (filter[i]=="stop") {
>                  run("Invert");
>           }
>         }
>         imageCalculator("AND create", "0","1");
>         imageCalculator("AND create", "Result of 0","2");
>         for (i=0;i<3;i++){
>           selectWindow(""+i);
>           close();
>         }
>         selectWindow("Result of 0");
>         close();
>         selectWindow("Result of Result of 0");
>         rename(a);
>         // Colour Thresholding------------
>         run("8-bit");
>         run("Fill Holes"); // Detected area inside green demarcated area
> has
> value 255
>         run("Invert"); // So invert (not invert LUT): Needed to exclude
> this
> area from calculations;
>
>         // Now obtain lumen part from image
>         selectImage(OriginalImage);
>         run("Duplicate...", "title=lumen_area");
>         LumenImage = getImageID();
>         selectImage(LumenImage);
>         a=getTitle();
>         run("RGBtoLab ");
>         run("RGB Stack");
>         run("Convert Stack to Images");
>         selectWindow("Red");
>         rename("0");
>         selectWindow("Green");
>         rename("1");
>         selectWindow("Blue");
>         rename("2");
>         min[0]=lumen_low_th;
>         max[0]=255;
>         filter[0]="pass";
>         min[1]=0;
>         max[1]=255;
>         filter[1]="pass";
>         min[2]=0;
>         max[2]=125;
>         filter[2]="pass";
>         for (i=0;i<3;i++){
>           selectWindow(""+i);
>           setThreshold(min[i], max[i]);
>           run("Make Binary", "thresholded remaining");
>           if (filter[i]=="stop")  run("Invert");
>         }
>         imageCalculator("AND create", "0","1");
>         imageCalculator("AND create", "Result of 0","2");
>         for (i=0;i<3;i++){
>           selectWindow(""+i);
>           close();
>         }
>         selectWindow("Result of 0");
>         close();
>         selectWindow("Result of Result of 0");
>         rename(a);
>         // Colour Thresholding------------
>         run("8-bit");
>         imageCalculator("AND", "lumen_area","exclude_area");
>
>         //Now obtain fibrosis component
>         selectImage(OriginalImage);
>         run("Duplicate...", "title=fibrosis_area");
>         FibrosisImage = getImageID();
>         selectImage(FibrosisImage);
>         a=getTitle();
>         run("RGBtoLab ");
>         run("RGB Stack");
>         run("Convert Stack to Images");
>         selectWindow("Red");
>         rename("0");
>         selectWindow("Green");
>         rename("1");
>         selectWindow("Blue");
>         rename("2");
>         min[0]=0;
>         max[0]=lumen_low_th-1;
>         filter[0]="pass";
>         //Based on discussion with Noura and Nynke
>         min[1]=fibrosis_high_th;
>         max[1]=255;
>         filter[1]="pass";
>         min[2]=0;
>         max[2]=255;
>         filter[2]="pass";
>         for (i=0;i<3;i++){
>           selectWindow(""+i);
>           setThreshold(min[i], max[i]);
>           run("Make Binary", "thresholded remaining");
>           if (filter[i]=="stop")  run("Invert");
>         }
>         imageCalculator("AND create", "0","1");
>         imageCalculator("AND create", "Result of 0","2");
>         for (i=0;i<3;i++){
>           selectWindow(""+i);
>           close();
>         }
>         selectWindow("Result of 0");
>         close();
>         selectWindow("Result of Result of 0");
>         rename(a);
>         // Colour Thresholding------------
>         run("8-bit");
>
>         // Obtain the remaining tissue compartments, in this case
> cardiomyocytes
>         selectImage("fibrosis_area");
>         run("Duplicate...", "title=cardiomyocytes_area");
>         CardioMyocyteImage = getImageID();
>         selectImage("fibrosis_area");
>         imageCalculator("AND", "fibrosis_area","exclude_area");
>         selectImage("cardiomyocytes_area");
>         run("Invert");
>         // Invert lumen and get rid of lumen in cardiomyocyte image
>         selectImage("lumen_area");
>         run("Invert");
>         imageCalculator("AND", "cardiomyocytes_area","lumen_area");
>         selectImage("lumen_area");
>         run("Invert");
>         selectImage("cardiomyocytes_area");
>         imageCalculator("AND", "cardiomyocytes_area","exclude_area");
>
>         // Now estimate percentages and present to user
>         // Everything with value zero will be measured as object!
>         run("Set Measurements...", "area limit redirect=None decimal=3");
> //
> General settings
>         // First Exclusion area
>         selectImage("exclude_area");
>         // We need the size of the exclusion part
>         totarea_exclusion=0;
>         run("Analyze Particles...", "size=0-Infinity circularity=0.00-1.00
> show=Nothing clear");
>         for (i=0; i<nResults; i++) {
>                         totarea_exclusion = totarea_exclusion+
> getResult(&quot;Area&quot;,i);
>                 }
>         //  lumen
>         selectImage(&quot;lumen_area&quot;);
>         run(&quot;Invert&quot;);
>         totarea_lumen=0;
>         run(&quot;Analyze Particles...&quot;, &quot;size=0-Infinity
> circularity=0.00-1.00 show=Nothing clear&quot;);
>         for (i=0; i&lt;nResults; i++) {
>                         totarea_lumen = totarea_lumen+
> getResult(&quot;Area&quot;,i);
>                 }
>         // Fibrosis
>         selectImage(&quot;fibrosis_area&quot;);
>         run(&quot;Invert&quot;);
>         totarea_fibrosis=0;
>         run(&quot;Analyze Particles...&quot;, &quot;size=0-Infinity
> circularity=0.00-1.00 show=Nothing clear&quot;);
>         for (i=0; i&lt;nResults; i++) {
>                         totarea_fibrosis = totarea_fibrosis+
> getResult(&quot;Area&quot;,i);
>                 }
>         // Cardiomyocytes
>         selectImage(&quot;cardiomyocytes_area&quot;);
>         run(&quot;Invert&quot;);
>         totarea_cardio=0;
>         run(&quot;Analyze Particles...&quot;, &quot;size=0-Infinity
> circularity=0.00-1.00 show=Nothing clear&quot;);
>         for (i=0; i&lt;nResults; i++) {
>                         totarea_cardio = totarea_cardio+
> getResult(&quot;Area&quot;,i);
>                 }
>         run(&quot;Clear Results&quot;);
>         setResult(&quot;Total area excluded (pixels)&quot;, 0,
> totarea_exclusion);
>         setResult(&quot;Total area Lumen (pixels)&quot;, 0, totarea_lumen);
>         setResult(&quot;Total area rest including cardiomyocytes
> (pixels)&quot;, 0, totarea_cardio);
>         setResult(&quot;Total area fibrosis (pixels)&quot;, 0,
> totarea_fibrosis);
>         updateResults();
>         selectWindow(&quot;Results&quot;);
>         run(&quot;Text...&quot;); // File>Save As>
> }// End of macro
>
>
>
> --
> View this message in context:
> http://imagej.1557.n6.nabble.com/Sirius-Red-staining-Macro-tp4999623.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

... [show rest of quote]

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[Gonzalo Bouchet]

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