requires("1.54s");

macro "Batch_DAPI_C1_MainNuclei_ManualThreshold_LessAggressive_withBatchReport_andAllAreas" {

    // =========================================================
    // DEFAULT PARAMETERS
    // =========================================================
    rollingBallNuc     = 25;
    gaussianSigmaNuc   = 0.8;

    // threshold iniziale, modificabile a mano per ogni immagine
    minThresholdNuc    = 20;
    maxThresholdNuc    = 255;

    minNucleusArea     = 40;
    minNucleusCirc     = 0.04;

    excludeEdges       = false;

    // default conservativo
    doWatershedNuclei  = false;

    saveOverlay        = true;
    fontSizeLabel      = 18;

    // =========================================================
    // SELECT FOLDERS
    // =========================================================
    inputDir = getDirectory("Scegli la cartella con le immagini C1");
    if (inputDir == "")
        exit("Nessuna cartella input.");

    outputDir = getDirectory("Scegli la cartella dove salvare i risultati");
    if (outputDir == "")
        exit("Nessuna cartella output.");

    if (!endsWith(inputDir, "/"))  inputDir  = inputDir  + "/";
    if (!endsWith(outputDir, "/")) outputDir = outputDir + "/";

    fileList = getFileList(inputDir);

    // =========================================================
    // PARAMETER DIALOG
    // =========================================================
    Dialog.create("Parametri nuclei batch");
    Dialog.addNumber("Rolling ball", rollingBallNuc);
    Dialog.addNumber("Gaussian sigma", gaussianSigmaNuc);
    Dialog.addNumber("Min threshold iniziale (8-bit)", minThresholdNuc);
    Dialog.addNumber("Max threshold iniziale (8-bit)", maxThresholdNuc);
    Dialog.addNumber("Min nucleus area", minNucleusArea);
    Dialog.addNumber("Min nucleus circularity", minNucleusCirc);
    Dialog.addCheckbox("Watershed nuclei", doWatershedNuclei);
    Dialog.addCheckbox("Exclude edge particles", excludeEdges);
    Dialog.addCheckbox("Save overlay", saveOverlay);
    Dialog.addNumber("Overlay font size", fontSizeLabel);
    Dialog.show();

    rollingBallNuc    = Dialog.getNumber();
    gaussianSigmaNuc  = Dialog.getNumber();
    minThresholdNuc   = Dialog.getNumber();
    maxThresholdNuc   = Dialog.getNumber();
    minNucleusArea    = Dialog.getNumber();
    minNucleusCirc    = Dialog.getNumber();
    doWatershedNuclei = Dialog.getCheckbox();
    excludeEdges      = Dialog.getCheckbox();
    saveOverlay       = Dialog.getCheckbox();
    fontSizeLabel     = Dialog.getNumber();

    // =========================================================
    // BATCH REPORTS
    // =========================================================
    batchReportCsv = outputDir + "Batch_Nuclei_Report.csv";
    batchAreaCsv   = outputDir + "Batch_Nuclei_All_Areas.csv";

    batchHeader =
        "Sample_ID," +
        "Count," +
        "Mean_area," +
        "SD_area," +
        "Min_area," +
        "Max_area," +
        "Total_area," +
        "Threshold_mode," +
        "Watershed_used," +
        "Exclude_edges\n";

    File.saveString(batchHeader, batchReportCsv);

    batchAreaHeader =
        "Sample_ID," +
        "Nucleus_ID," +
        "Area," +
        "X," +
        "Y\n";

    File.saveString(batchAreaHeader, batchAreaCsv);

    // =========================================================
    // LOOP FILES
    // =========================================================
    for (f = 0; f < fileList.length; f++) {

        name = fileList[f];
        path = inputDir + name;

        if (File.isDirectory(path))
            continue;

        if (!(endsWith(name, "C1.tif") || endsWith(name, "C1.tiff") ||
              endsWith(name, "C1.TIF") || endsWith(name, "C1.TIFF")))
            continue;

        open(path);
        originalTitle = getTitle();

        run("Select None");
        roiManager("Reset");

        // -----------------------------------------------------
        // BASE NAME
        // -----------------------------------------------------
        baseName = originalTitle;

        if (endsWith(baseName, ".tif") || endsWith(baseName, ".TIF"))
            baseName = substring(baseName, 0, lengthOf(baseName) - 4);
        else if (endsWith(baseName, ".tiff") || endsWith(baseName, ".TIFF"))
            baseName = substring(baseName, 0, lengthOf(baseName) - 5);

        roiZip     = outputDir + baseName + "_Nuclei_ROIs.zip";
        areaCsv    = outputDir + baseName + "_Nuclei_Area.csv";
        summaryCsv = outputDir + baseName + "_Nuclei_Summary.csv";
        overlayTif = outputDir + baseName + "_Nuclei_Overlay.tif";

        // =====================================================
        // PREP IMAGE
        // =====================================================
        Stack.getDimensions(w, h, c, z, t);

        if (z > 1) {
            run("Z Project...", "projection=[Max Intensity]");
            rename("DAPI_MAX");
            selectWindow(originalTitle);
            close();
        } else {
            run("Duplicate...", "title=DAPI_MAX");
            selectWindow(originalTitle);
            close();
        }

        // copia per overlay finale
        selectWindow("DAPI_MAX");
        run("Select None");
        run("Duplicate...", "title=DAPI_FOR_OVERLAY");

        // =====================================================
        // NUCLEI SEGMENTATION
        // =====================================================
        selectWindow("DAPI_MAX");
        run("Select None");
        run("Duplicate...", "title=NUC_PREP");

        selectWindow("NUC_PREP");
        run("8-bit");
        run("Subtract Background...", "rolling=" + rollingBallNuc);

        if (gaussianSigmaNuc > 0)
            run("Gaussian Blur...", "sigma=" + gaussianSigmaNuc);

        run("Enhance Contrast...", "saturated=0.35 normalize");

        // threshold iniziale, modificabile a mano
        setThreshold(minThresholdNuc, maxThresholdNuc);
        run("Threshold...");

        waitForUser("[" + baseName + "]\nControlla la sogliatura dei nuclei.\nRegola la threshold se necessario.\nNON premere Apply.\nPremi solo OK qui per continuare.");

        selectWindow("NUC_PREP");
        setOption("BlackBackground", false);
        run("Convert to Mask");

        // Pulizia minima per non spezzare nuclei singoli
        run("Fill Holes");

        if (doWatershedNuclei)
            run("Watershed");

        rename("NUCLEI_MASK");

        // =====================================================
        // ANALYZE PARTICLES
        // =====================================================
        roiManager("Reset");

        if (isOpen("Results")) {
            selectWindow("Results");
            run("Clear Results");
        }

        run("Set Measurements...", "area centroid shape decimal=3");

        options = "size=" + minNucleusArea + "-Infinity circularity=" + minNucleusCirc + "-1.00 display add";
        if (excludeEdges)
            options = options + " exclude";

        run("Analyze Particles...", options);
        nucleiCount = roiManager("count");

        // =====================================================
        // NO NUCLEI FOUND
        // =====================================================
        if (nucleiCount == 0) {

            if (isOpen("Results")) {
                selectWindow("Results");
                run("Clear Results");
            }

            setResult("Image", 0, baseName);
            setResult("Count", 0, 0);
            setResult("Mean_area", 0, 0);
            setResult("SD_area", 0, 0);
            setResult("Min_area", 0, 0);
            setResult("Max_area", 0, 0);
            setResult("Total_area", 0, 0);
            setResult("Threshold_mode", 0, "manual_per_image");
            setResult("Watershed_used", 0, doWatershedNuclei);
            setResult("Exclude_edges", 0, excludeEdges);
            updateResults();
            saveAs("Results", summaryCsv);

            batchLine =
                baseName + "," +
                "0,0,0,0,0,0," +
                "manual_per_image," +
                doWatershedNuclei + "," +
                excludeEdges + "\n";
            File.append(batchLine, batchReportCsv);

            close("*");
            roiManager("Reset");
            run("Collect Garbage");
            continue;
        }

        // =====================================================
        // SAVE ROIs
        // =====================================================
        roiManager("Deselect");
        roiManager("Save", roiZip);

        // =====================================================
        // MEASURE EACH NUCLEUS
        // =====================================================
        if (isOpen("Results")) {
            selectWindow("Results");
            run("Clear Results");
        }

        nucArea   = newArray(nucleiCount);
        nucX      = newArray(nucleiCount);
        nucY      = newArray(nucleiCount);
        totalArea = 0;

        run("Set Measurements...", "area centroid shape decimal=3");

        for (i = 0; i < nucleiCount; i++) {
            selectWindow("DAPI_MAX");
            roiManager("select", i);
            run("Measure");
            nucArea[i] = getResult("Area", i);
            nucX[i]    = getResult("X", i);
            nucY[i]    = getResult("Y", i);
            totalArea += nucArea[i];
        }

        if (nucleiCount == 1) {
            minA  = nucArea[0];
            maxA  = nucArea[0];
            meanA = nucArea[0];
            sdA   = 0;
        } else {
            Array.getStatistics(nucArea, minA, maxA, meanA, sdA);
            if (sdA != sdA) sdA = 0;
        }

        // =====================================================
        // APPEND ALL SINGLE NUCLEI TO GLOBAL BATCH AREA FILE
        // =====================================================
        for (i = 0; i < nucleiCount; i++) {
            line =
                baseName + "," +
                (i + 1) + "," +
                d2s(nucArea[i], 6) + "," +
                d2s(nucX[i], 3) + "," +
                d2s(nucY[i], 3) + "\n";

            File.append(line, batchAreaCsv);
        }

        // =====================================================
        // SAVE AREA CSV
        // =====================================================
        if (isOpen("Results")) {
            selectWindow("Results");
            run("Clear Results");
        }

        for (i = 0; i < nucleiCount; i++) {
            setResult("Image", i, baseName);
            setResult("Nucleus_ID", i, i + 1);
            setResult("Area", i, nucArea[i]);
            setResult("X", i, nucX[i]);
            setResult("Y", i, nucY[i]);
        }

        updateResults();
        saveAs("Results", areaCsv);

        // =====================================================
        // SAVE SUMMARY CSV
        // =====================================================
        if (isOpen("Results")) {
            selectWindow("Results");
            run("Clear Results");
        }

        setResult("Image", 0, baseName);
        setResult("Count", 0, nucleiCount);
        setResult("Mean_area", 0, meanA);
        setResult("SD_area", 0, sdA);
        setResult("Min_area", 0, minA);
        setResult("Max_area", 0, maxA);
        setResult("Total_area", 0, totalArea);
        setResult("Threshold_mode", 0, "manual_per_image");
        setResult("Watershed_used", 0, doWatershedNuclei);
        setResult("Exclude_edges", 0, excludeEdges);
        updateResults();
        saveAs("Results", summaryCsv);

        // =====================================================
        // APPEND TO BATCH REPORT
        // =====================================================
        batchLine =
            baseName + "," +
            nucleiCount + "," +
            d2s(meanA, 6) + "," +
            d2s(sdA, 6) + "," +
            d2s(minA, 6) + "," +
            d2s(maxA, 6) + "," +
            d2s(totalArea, 6) + "," +
            "manual_per_image," +
            doWatershedNuclei + "," +
            excludeEdges + "\n";

        File.append(batchLine, batchReportCsv);

        // =====================================================
        // SAVE OVERLAY IMAGE
        // =====================================================
        if (saveOverlay) {
            selectWindow("DAPI_FOR_OVERLAY");
            run("RGB Color");
            rename("OVERLAY_RGB");

            setForegroundColor(255, 255, 0);
            setLineWidth(1);

            for (i = 0; i < roiManager("count"); i++) {
                selectWindow("OVERLAY_RGB");
                roiManager("select", i);
                run("Draw");
            }

            setForegroundColor(255, 255, 0);
            setFont("SansSerif", fontSizeLabel, "antialiased");

            for (i = 0; i < nucleiCount; i++) {
                drawString("" + (i + 1), nucX[i] + 4, nucY[i] - 4);
            }

            drawString("Nuclei = " + nucleiCount, 20, 30);
            drawString("Threshold = manual", 20, 55);

            saveAs("Tiff", overlayTif);
            close();
        }

        // =====================================================
        // CLEANUP
        // =====================================================
        close("*");
        roiManager("Reset");
        run("Collect Garbage");
    }

    print("=== Batch nuclei principali completato ===");
    print("Batch report salvato in: " + batchReportCsv);
    print("Batch aree singoli nuclei salvato in: " + batchAreaCsv);
}