requires("1.54s");

macro "Batch_DAPI_C1_Micronuclei_FixedThreshold_40_255_Area3_30_Circ0p5_NoOverlapMainNuclei" {

    // =========================================================
    // DEFAULT PARAMETERS
    // =========================================================
    rollingBallNuc      = 15;
    gaussianSigmaNuc    = 0.6;

    // fixed threshold
    minThresholdNuc     = 50;
    maxThresholdNuc     = 255;

    // micronuclei filters
    minMicronucleusArea = 2;
    maxMicronucleusArea = 30;
    minMicronucleusCirc = 0.3;

    excludeEdges        = true;
    saveOverlay         = true;
    fontSizeLabel       = 16;

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

    mainRoiDir = getDirectory("Scegli la cartella con le ROI dei nuclei principali");
    if (mainRoiDir == "")
        exit("Nessuna cartella ROI nuclei principali.");

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

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

    fileList = getFileList(inputDir);

    // =========================================================
    // PARAMETER DIALOG
    // =========================================================
    Dialog.create("Parametri micronuclei batch");
    Dialog.addNumber("Rolling ball", rollingBallNuc);
    Dialog.addNumber("Gaussian sigma", gaussianSigmaNuc);
    Dialog.addNumber("Min threshold fixed (8-bit)", minThresholdNuc);
    Dialog.addNumber("Max threshold fixed (8-bit)", maxThresholdNuc);
    Dialog.addNumber("Min micronucleus area", minMicronucleusArea);
    Dialog.addNumber("Max micronucleus area", maxMicronucleusArea);
    Dialog.addNumber("Min micronucleus circularity", minMicronucleusCirc);
    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();
    minMicronucleusArea = Dialog.getNumber();
    maxMicronucleusArea = Dialog.getNumber();
    minMicronucleusCirc = Dialog.getNumber();
    excludeEdges        = Dialog.getCheckbox();
    saveOverlay         = Dialog.getCheckbox();
    fontSizeLabel       = Dialog.getNumber();

    // =========================================================
    // BATCH FILES
    // =========================================================
    batchSummaryCsv = outputDir + "Batch_Micronuclei_Report.csv";
    batchAllCsv     = outputDir + "Batch_Micronuclei_All.csv";

    batchSummaryHeader =
        "Sample_ID," +
        "Main_nuclei_count," +
        "Micronuclei_count," +
        "Micronuclei_per_main_nucleus," +
        "Mean_area," +
        "SD_area," +
        "Min_area," +
        "Max_area," +
        "Total_area," +
        "Threshold_mode," +
        "Min_threshold_fixed," +
        "Max_threshold_fixed," +
        "Min_area_filter," +
        "Max_area_filter," +
        "Min_circularity_filter," +
        "Exclude_edges\n";

    File.saveString(batchSummaryHeader, batchSummaryCsv);

    batchAllHeader =
        "Sample_ID," +
        "Micronucleus_ID," +
        "Area," +
        "X," +
        "Y\n";

    File.saveString(batchAllHeader, batchAllCsv);

    // =========================================================
    // 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);

        mainRoiZip = mainRoiDir + baseName + "_Nuclei_ROIs.zip";

        if (!File.exists(mainRoiZip)) {
            print("ROI nuclei principali non trovate per: " + baseName);
            close();
            continue;
        }

        roiZip     = outputDir + baseName + "_Micronuclei_ROIs.zip";
        areaCsv    = outputDir + baseName + "_Micronuclei_Area.csv";
        summaryCsv = outputDir + baseName + "_Micronuclei_Summary.csv";
        overlayTif = outputDir + baseName + "_Micronuclei_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();
        }

        selectWindow("DAPI_MAX");
        run("Select None");
        run("Duplicate...", "title=DAPI_FOR_OVERLAY");

        // =====================================================
        // LOAD MAIN NUCLEI ROIs
        // =====================================================
        roiManager("Reset");
        roiManager("Open", mainRoiZip);
        mainNucleiCount = roiManager("count");

        if (mainNucleiCount == 0) {
            print("Nessuna ROI nucleo principale in: " + mainRoiZip);
            close("*");
            roiManager("Reset");
            continue;
        }

        // =====================================================
        // SEGMENT MICRONUCLEI CANDIDATES
        // =====================================================
        selectWindow("DAPI_MAX");
        run("Select None");
        run("Duplicate...", "title=MICRO_PREP");

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

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

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

        setThreshold(minThresholdNuc, maxThresholdNuc);
        setOption("BlackBackground", false);
        run("Convert to Mask");
        run("Fill Holes");
        rename("MICRONUCLEI_MASK");

        // =====================================================
        // FIND ALL CANDIDATES
        // =====================================================
        roiManager("Reset");
        if (isOpen("Results")) {
            selectWindow("Results");
            run("Clear Results");
        }

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

        options = "size=" + minMicronucleusArea + "-" + maxMicronucleusArea +
                  " circularity=" + minMicronucleusCirc + "-1.00 display add";
        if (excludeEdges)
            options = options + " exclude";

        selectWindow("MICRONUCLEI_MASK");
        run("Analyze Particles...", options);

        candCount = roiManager("count");

        if (candCount == 0) {

            micronucleiPerMainNucleus = 0;

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

            setResult("Image", 0, baseName);
            setResult("Main_nuclei_count", 0, mainNucleiCount);
            setResult("Micronuclei_count", 0, 0);
            setResult("Micronuclei_per_main_nucleus", 0, micronucleiPerMainNucleus);
            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, "fixed");
            setResult("Min_threshold_fixed", 0, minThresholdNuc);
            setResult("Max_threshold_fixed", 0, maxThresholdNuc);
            setResult("Min_area_filter", 0, minMicronucleusArea);
            setResult("Max_area_filter", 0, maxMicronucleusArea);
            setResult("Min_circularity_filter", 0, minMicronucleusCirc);
            setResult("Exclude_edges", 0, excludeEdges);
            updateResults();
            saveAs("Results", summaryCsv);

            batchLine =
                baseName + "," +
                mainNucleiCount + "," +
                "0," +
                "0," +
                "0,0,0,0,0," +
                "fixed," +
                minThresholdNuc + "," +
                maxThresholdNuc + "," +
                minMicronucleusArea + "," +
                maxMicronucleusArea + "," +
                d2s(minMicronucleusCirc, 3) + "," +
                excludeEdges + "\n";
            File.append(batchLine, batchSummaryCsv);

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

        // =====================================================
        // STORE CANDIDATE ROIs AS TEMP ZIP
        // =====================================================
        tempCandZip = outputDir + "__temp_candidates_" + baseName + ".zip";
        roiManager("Deselect");
        roiManager("Save", tempCandZip);

        // =====================================================
        // MEASURE CANDIDATES
        // =====================================================
        candArea = newArray(candCount);
        candX    = newArray(candCount);
        candY    = newArray(candCount);
        keepFlag = newArray(candCount);

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

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

        for (i = 0; i < candCount; i++) {
            selectWindow("DAPI_MAX");
            roiManager("select", i);
            run("Measure");
            candArea[i] = getResult("Area", i);
            candX[i]    = getResult("X", i);
            candY[i]    = getResult("Y", i);
            keepFlag[i] = 1;
        }

        // =====================================================
        // FILTER BY TRUE GEOMETRIC OVERLAP
        // =====================================================
        for (i = 0; i < candCount; i++) {

            overlapFound = 0;

            roiManager("Reset");
            roiManager("Open", tempCandZip);
            roiManager("select", i);

            roiManager("Add");
            candSelIndex = roiManager("count") - 1;

            roiManager("Open", mainRoiZip);
            totalNow = roiManager("count");

            for (j = candSelIndex + 1; j < totalNow; j++) {

                roiManager("select", newArray(candSelIndex, j));
                roiManager("AND");

                if (selectionType() != -1) {
                    getStatistics(andArea);
                    if (andArea > 0) {
                        overlapFound = 1;
                        break;
                    }
                }
            }

            if (overlapFound == 1)
                keepFlag[i] = 0;
        }

        // =====================================================
        // COUNT FINAL KEPT MICRONUCLEI
        // =====================================================
        micronucleiCount = 0;
        for (i = 0; i < candCount; i++) {
            if (keepFlag[i] == 1)
                micronucleiCount++;
        }

        if (mainNucleiCount > 0)
            micronucleiPerMainNucleus = micronucleiCount / mainNucleiCount;
        else
            micronucleiPerMainNucleus = 0;

        if (micronucleiCount == 0) {

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

            setResult("Image", 0, baseName);
            setResult("Main_nuclei_count", 0, mainNucleiCount);
            setResult("Micronuclei_count", 0, 0);
            setResult("Micronuclei_per_main_nucleus", 0, micronucleiPerMainNucleus);
            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, "fixed");
            setResult("Min_threshold_fixed", 0, minThresholdNuc);
            setResult("Max_threshold_fixed", 0, maxThresholdNuc);
            setResult("Min_area_filter", 0, minMicronucleusArea);
            setResult("Max_area_filter", 0, maxMicronucleusArea);
            setResult("Min_circularity_filter", 0, minMicronucleusCirc);
            setResult("Exclude_edges", 0, excludeEdges);
            updateResults();
            saveAs("Results", summaryCsv);

            batchLine =
                baseName + "," +
                mainNucleiCount + "," +
                "0," +
                d2s(micronucleiPerMainNucleus, 6) + "," +
                "0,0,0,0,0," +
                "fixed," +
                minThresholdNuc + "," +
                maxThresholdNuc + "," +
                minMicronucleusArea + "," +
                maxMicronucleusArea + "," +
                d2s(minMicronucleusCirc, 3) + "," +
                excludeEdges + "\n";
            File.append(batchLine, batchSummaryCsv);

            roiManager("Reset");
            File.delete(tempCandZip);
            close("*");
            run("Collect Garbage");
            continue;
        }

        // =====================================================
        // BUILD FINAL ARRAYS
        // =====================================================
        microArea = newArray(micronucleiCount);
        microX    = newArray(micronucleiCount);
        microY    = newArray(micronucleiCount);

        totalArea = 0;
        k = 0;

        for (i = 0; i < candCount; i++) {
            if (keepFlag[i] == 1) {
                microArea[k] = candArea[i];
                microX[k]    = candX[i];
                microY[k]    = candY[i];
                totalArea   += microArea[k];
                k++;
            }
        }

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

        // =====================================================
        // REBUILD FINAL ROI SET AND SAVE
        // =====================================================
        roiManager("Reset");
        roiManager("Open", tempCandZip);

        for (i = candCount - 1; i >= 0; i--) {
            if (keepFlag[i] == 0) {
                roiManager("select", i);
                roiManager("Delete");
            }
        }

        roiManager("Deselect");
        roiManager("Save", roiZip);

        // =====================================================
        // APPEND TO GLOBAL ALL CSV
        // =====================================================
        for (i = 0; i < micronucleiCount; i++) {
            line =
                baseName + "," +
                (i + 1) + "," +
                d2s(microArea[i], 6) + "," +
                d2s(microX[i], 3) + "," +
                d2s(microY[i], 3) + "\n";

            File.append(line, batchAllCsv);
        }

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

        for (i = 0; i < micronucleiCount; i++) {
            setResult("Image", i, baseName);
            setResult("Micronucleus_ID", i, i + 1);
            setResult("Area", i, microArea[i]);
            setResult("X", i, microX[i]);
            setResult("Y", i, microY[i]);
        }

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

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

        setResult("Image", 0, baseName);
        setResult("Main_nuclei_count", 0, mainNucleiCount);
        setResult("Micronuclei_count", 0, micronucleiCount);
        setResult("Micronuclei_per_main_nucleus", 0, micronucleiPerMainNucleus);
        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, "fixed");
        setResult("Min_threshold_fixed", 0, minThresholdNuc);
        setResult("Max_threshold_fixed", 0, maxThresholdNuc);
        setResult("Min_area_filter", 0, minMicronucleusArea);
        setResult("Max_area_filter", 0, maxMicronucleusArea);
        setResult("Min_circularity_filter", 0, minMicronucleusCirc);
        setResult("Exclude_edges", 0, excludeEdges);
        updateResults();
        saveAs("Results", summaryCsv);

        // =====================================================
        // APPEND TO BATCH SUMMARY
        // =====================================================
        batchLine =
            baseName + "," +
            mainNucleiCount + "," +
            micronucleiCount + "," +
            d2s(micronucleiPerMainNucleus, 6) + "," +
            d2s(meanA, 6) + "," +
            d2s(sdA, 6) + "," +
            d2s(minA, 6) + "," +
            d2s(maxA, 6) + "," +
            d2s(totalArea, 6) + "," +
            "fixed," +
            minThresholdNuc + "," +
            maxThresholdNuc + "," +
            minMicronucleusArea + "," +
            maxMicronucleusArea + "," +
            d2s(minMicronucleusCirc, 3) + "," +
            excludeEdges + "\n";

        File.append(batchLine, batchSummaryCsv);

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

            roiManager("Reset");
            roiManager("Open", mainRoiZip);
            roiManager("Deselect");
            roiManager("Set Color", "magenta");
            roiManager("Show All");
            run("Flatten");
            rename("OVERLAY_MAIN");

            roiManager("Reset");
            roiManager("Open", roiZip);
            selectWindow("OVERLAY_MAIN");
            roiManager("Deselect");
            roiManager("Set Color", "cyan");
            roiManager("Show All");
            run("Flatten");
            rename("OVERLAY_FINAL");

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

            for (i = 0; i < micronucleiCount; i++) {
                drawString("" + (i + 1), microX[i] + 3, microY[i] - 3);
            }

            drawString("Micronuclei = " + micronucleiCount, 20, 30);
            drawString("Micronuclei / main nucleus = " + d2s(micronucleiPerMainNucleus, 3), 20, 55);
            drawString("Thr = " + minThresholdNuc + "-" + maxThresholdNuc, 20, 80);
            drawString("Area = " + minMicronucleusArea + "-" + maxMicronucleusArea + " ; Circ >= " + d2s(minMicronucleusCirc, 2), 20, 105);

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

            if (isOpen("OVERLAY_MAIN")) {
                selectWindow("OVERLAY_MAIN");
                close();
            }
        }

        // =====================================================
        // CLEANUP
        // =====================================================
        roiManager("Reset");
        File.delete(tempCandZip);
        close("*");
        run("Collect Garbage");
    }

    print("=== Batch micronuclei completato ===");
    print("Batch summary salvato in: " + batchSummaryCsv);
    print("Batch per micronucleo salvato in: " + batchAllCsv);
}