requires("1.54p");

macro "Batch_C2_Phalloidin_AreaPerNucleus_v9_FixedThreshold_Conservative_WithBatchSummary" {

    // =========================================================
    // DEFAULT PARAMETERS
    // =========================================================
    rollingBallCell   = 80;
    gaussianSigmaCell = 1.0;
    minThreshold      = 15;
    maxThreshold      = 255;
    minPhalloidinArea = 200;
    excludeEdges      = false;
    saveOverlay       = true;
    fontSizeLabel     = 18;

    // =========================================================
    // SELECT FOLDERS
    // =========================================================
    c2Dir = getDirectory("Cartella immagini C2 falloidina");
    if (c2Dir == "")
        exit("Nessuna cartella C2 selezionata.");

    roiDir = getDirectory("Cartella ROI nuclei");
    if (roiDir == "")
        exit("Nessuna cartella ROI selezionata.");

    outDir = getDirectory("Cartella output");
    if (outDir == "")
        exit("Nessuna cartella output selezionata.");

    if (!endsWith(c2Dir, "/")) c2Dir = c2Dir + "/";
    if (!endsWith(roiDir, "/")) roiDir = roiDir + "/";
    if (!endsWith(outDir, "/")) outDir = outDir + "/";

    // =========================================================
    // PARAMETER DIALOG
    // =========================================================
    Dialog.create("C2 falloidina - area per nucleo");
    Dialog.addNumber("Rolling ball", rollingBallCell);
    Dialog.addNumber("Gaussian sigma", gaussianSigmaCell);
    Dialog.addNumber("Min threshold (fixed)", minThreshold);
    Dialog.addNumber("Max threshold (fixed)", maxThreshold);
    Dialog.addNumber("Min phalloidin particle area", minPhalloidinArea);
    Dialog.addCheckbox("Exclude edge particles", excludeEdges);
    Dialog.addCheckbox("Save overlay", saveOverlay);
    Dialog.addNumber("Overlay font size", fontSizeLabel);
    Dialog.show();

    rollingBallCell   = Dialog.getNumber();
    gaussianSigmaCell = Dialog.getNumber();
    minThreshold      = Dialog.getNumber();
    maxThreshold      = Dialog.getNumber();
    minPhalloidinArea = Dialog.getNumber();
    excludeEdges      = Dialog.getCheckbox();
    saveOverlay       = Dialog.getCheckbox();
    fontSizeLabel     = Dialog.getNumber();

    fileList = getFileList(c2Dir);

    // =========================================================
    // PREPARE BATCH SUMMARY CSV
    // =========================================================
    batchSummaryCsv = outDir + "Batch_C2_PhalloidinArea_per_nucleus_summary.csv";

    batchHeader =
        "Sample_ID," +
        "Phalloidin_area_per_nucleus," +
        "Phalloidin_SD_area_divided_by_nuclei," +
        "Nuclei_count," +
        "Phalloidin_particles," +
        "Phalloidin_total_area," +
        "Phalloidin_mean_area," +
        "Phalloidin_SD_area," +
        "Phalloidin_CV_area," +
        "Phalloidin_min_area," +
        "Phalloidin_max_area," +
        "Min_threshold_fixed," +
        "Max_threshold_fixed\n";

    File.saveString(batchHeader, batchSummaryCsv);

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

        name = fileList[f];
        path = c2Dir + name;

        if (File.isDirectory(path))
            continue;

        if (!(endsWith(name, "_C2.tif") || endsWith(name, "_C2.tiff") ||
              endsWith(name, "_C2.TIF") || endsWith(name, "_C2.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);

        if (endsWith(baseName, "_C2"))
            sampleBase = substring(baseName, 0, lengthOf(baseName) - 3);
        else
            sampleBase = baseName;

        roiZip = roiDir + sampleBase + "_C1_Nuclei_ROIs.zip";

        if (!File.exists(roiZip)) {
            print("ROI nuclei non trovate: " + sampleBase);
            close();
            continue;
        }

        summaryCsv       = outDir + sampleBase + "_C2_PhalloidinArea_per_nucleus.csv";
        overlayTif       = outDir + sampleBase + "_C2_PhalloidinMask_with_Nuclei.tif";
        phalloidinRoiZip = outDir + sampleBase + "_C2_Phalloidin_ROIs.zip";

        // -----------------------------------------------------
        // PREPARE IMAGE
        // -----------------------------------------------------
        Stack.getDimensions(w, h, c, z, t);

        if (z > 1) {
            run("Z Project...", "projection=[Max Intensity]");
            rename("C2_MAX");
            selectWindow(originalTitle);
            close();
        } else {
            rename("C2_MAX");
        }

        selectWindow("C2_MAX");
        run("Select None");
        run("Duplicate...", "title=C2_OVERLAY_BASE");

        // -----------------------------------------------------
        // COUNT NUCLEI FROM ROI ZIP
        // -----------------------------------------------------
        roiManager("Reset");
        roiManager("Open", roiZip);
        nucleiCount = roiManager("count");

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

        // -----------------------------------------------------
        // SEGMENT PHALLOIDIN - CONSERVATIVE
        // -----------------------------------------------------
        selectWindow("C2_MAX");
        run("Select None");
        run("Duplicate...", "title=C2_SEG");

        selectWindow("C2_SEG");
        run("8-bit");
        run("Subtract Background...", "rolling=" + rollingBallCell);

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

        run("Enhance Contrast...", "saturated=1.0 normalize");

        setThreshold(minThreshold, maxThreshold);
        setOption("BlackBackground", false);
        run("Convert to Mask");

        // Pulizia leggera, più conservativa delle operazioni morfologiche
        run("Despeckle");

        rename("PHALLOIDIN_MASK");

        // -----------------------------------------------------
        // MEASURE TOTAL PHALLOIDIN AREA + SAVE PHALLOIDIN ROIs
        // -----------------------------------------------------
        roiManager("Reset");
        if (isOpen("Results")) {
            selectWindow("Results");
            run("Clear Results");
        }

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

        options = "size=" + minPhalloidinArea + "-Infinity circularity=0.00-1.00 display clear add";
        if (excludeEdges)
            options = options + " exclude";

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

        nParts = nResults;
        totalPhalloidinArea = 0;
        areas = newArray(nParts);

        for (i = 0; i < nParts; i++) {
            a = getResult("Area", i);
            totalPhalloidinArea += a;
            areas[i] = a;
        }

        if (nParts > 0) {
            Array.getStatistics(areas, minA, maxA, meanA, sdA);
            roiManager("Deselect");
            roiManager("Save", phalloidinRoiZip);
        } else {
            minA = 0;
            maxA = 0;
            meanA = 0;
            sdA = 0;
        }

        phalloidinAreaPerNucleus = totalPhalloidinArea / nucleiCount;
        sdAreaDividedByNuclei = sdA / nucleiCount;

        if (meanA > 0)
            cvArea = sdA / meanA;
        else
            cvArea = 0;

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

        setResult("Sample_ID", 0, sampleBase);
        setResult("Phalloidin_area_per_nucleus", 0, phalloidinAreaPerNucleus);
        setResult("Phalloidin_SD_area_divided_by_nuclei", 0, sdAreaDividedByNuclei);
        setResult("Nuclei_count", 0, nucleiCount);

        setResult("Phalloidin_particles", 0, nParts);
        setResult("Phalloidin_total_area", 0, totalPhalloidinArea);
        setResult("Phalloidin_mean_area", 0, meanA);
        setResult("Phalloidin_SD_area", 0, sdA);
        setResult("Phalloidin_CV_area", 0, cvArea);
        setResult("Phalloidin_min_area", 0, minA);
        setResult("Phalloidin_max_area", 0, maxA);
        setResult("Min_threshold_fixed", 0, minThreshold);
        setResult("Max_threshold_fixed", 0, maxThreshold);

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

        // -----------------------------------------------------
        // APPEND TO BATCH SUMMARY CSV
        // -----------------------------------------------------
        batchLine =
            sampleBase + "," +
            d2s(phalloidinAreaPerNucleus, 6) + "," +
            d2s(sdAreaDividedByNuclei, 6) + "," +
            nucleiCount + "," +
            nParts + "," +
            d2s(totalPhalloidinArea, 6) + "," +
            d2s(meanA, 6) + "," +
            d2s(sdA, 6) + "," +
            d2s(cvArea, 6) + "," +
            d2s(minA, 6) + "," +
            d2s(maxA, 6) + "," +
            minThreshold + "," +
            maxThreshold + "\n";

        File.append(batchLine, batchSummaryCsv);

        // -----------------------------------------------------
        // SAVE OVERLAY WITH PHALLOIDIN ROI + NUCLEI
        // -----------------------------------------------------
        if (saveOverlay) {

            // base image
            selectWindow("C2_OVERLAY_BASE");
            run("RGB Color");

            // show phalloidin ROIs in green
            roiManager("Reset");
            if (File.exists(phalloidinRoiZip))
                roiManager("Open", phalloidinRoiZip);

            roiManager("Deselect");
            roiManager("Set Color", "green");
            roiManager("Show All");
            run("Flatten");
            rename("OVERLAY_PHALLOIDIN");

            // add nuclei in magenta
            roiManager("Reset");
            roiManager("Open", roiZip);

            selectWindow("OVERLAY_PHALLOIDIN");
            roiManager("Deselect");
            roiManager("Set Color", "magenta");
            roiManager("Show All with labels");
            run("Flatten");
            rename("FINAL_OVERLAY");

            // add yellow text
            selectWindow("FINAL_OVERLAY");
            setForegroundColor(255, 255, 0);
            setFont("SansSerif", fontSizeLabel, "antialiased");
            drawString("Area/nucleus = " + d2s(phalloidinAreaPerNucleus, 2), 20, 30);
            drawString("SD/nuclei = " + d2s(sdAreaDividedByNuclei, 2), 20, 55);
            drawString("Nuclei = " + nucleiCount, 20, 80);
            drawString("CV = " + d2s(cvArea, 3), 20, 105);

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

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

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

    print("=== Batch C2 phalloidin area per nucleus completato ===");
    print("Summary batch salvato in: " + batchSummaryCsv);
}