Update file ScratchAssay.ijm

*/

/*

defaultFiducialPlacementOption = 1;  // 0 if the fiducials are placed along the gap. 1 if they are placed to the sides of it.

defaultGapOrientationOption = 0;  // 0 if the gap is vertical. 1 if the gap is horizontal. 2 if the gap orientation is unknown or varies from series to series.

fiducialBlurFactor = 1/46;  // The higher fiducialBlurFactor is, the more blurred the fiducial detection image will be. (Gaussian blur sigma will be equal to image width * fiducialBlurFactor.)

defaultContrastEnhancementPercentage = 20;

selectionColor = "red";

roiManagerWindowPadding = 10;  // Padding between the ROI Manager window and the edge of the screen (pixels).

roiManagerWindowWidth = 500;  // Width of the ROI Manager window (pixels).

roiManagerWindowHeight = 600;  // Height of the ROI Manager window (pixels).

logWindowPaddding = 100;  // Padding between the Log window and the edge of the screen (pixels).

resultsWindowPaddding = 200;  // Padding between the Results window and the edge of the screen (pixels).

intermediateDebugImagePrefix = "__";

intermediateSignificantImagePrefix = "_";

fiducialPlacementOptions = newArray("along the gap", "across the gap");

gapOrientationOptions = newArray("vertical", "horizontal", "unknown / varies");

minContrastEnhancementPercentage = 0;

maxContrastEnhancementPercentage = 90;

////////////////////////////////////////////////////////////////////////////////

		close(intermediateDebugImagePrefix + "*");

		close(intermediateSignificantImagePrefix + "*");

	}

	roiManager("reset");

	close("ROI Manager");

	close(summaryTitle);

	// Set the size and position of the Log window.

	selectIfExists("Log");

	Table.setLocationAndSize(logWindowPaddding, logWindowPaddding, screenWidth - logWindowPaddding * 2, screenHeight - logWindowPaddding * 2, "Log");

	// Set the size and position of the ROI manager window.

	roiManager("reset");

	run("ROI Manager...");

	selectIfExists("ROI Manager");

	Table.setLocationAndSize(screenWidth - roiManagerWindowPadding - roiManagerWindowWidth, roiManagerWindowPadding, roiManagerWindowWidth, roiManagerWindowHeight, "ROI Manager");

}

  	Dialog.addRadioButtonGroup("Fiducial Placement", fiducialPlacementOptions, 1, 2, fiducialPlacementOptions[defaultFiducialPlacementOption]);

	Dialog.setInsets(10, 10, 0);

	Dialog.addNumber("Contrast enhancement (" + minContrastEnhancementPercentage + ".." + maxContrastEnhancementPercentage + ", default " + defaultContrastEnhancementPercentage + "):", defaultContrastEnhancementPercentage, 1, 5, "");

	Dialog.setInsets(10, 0, 0);

	Dialog.addMessage("------------------------------------------------------------------------------------------------------------------------------------------------");

	fiducialsAreAlongTheGap = (Dialog.getRadioButton() == fiducialPlacementOptions[0]);

	print("fiducialsAreAlongTheGap: " + fiducialsAreAlongTheGap);

	// Read the saturation percentage for contrast enhancement.

	contrastEnhancementPercentage = Math.max(Math.min(Dialog.getNumber(), maxContrastEnhancementPercentage), minContrastEnhancementPercentage);

	print("contrastEnhancementPercentage: " + contrastEnhancementPercentage);

	// Read the debugging options.

	useBatchModeForComputations = Dialog.getCheckbox();

	// Close older versions of the intermediate images (unless they should be kept).

	if (Table.columnExists("Group")) {

		Table.deleteColumn("Group");

	}	

	// Set the size and position of the Results window.

	Table.setLocationAndSize(resultsWindowPaddding, resultsWindowPaddding, screenWidth - resultsWindowPaddding * 2, screenHeight - resultsWindowPaddding * 2, "Results");

	/*

	Table.renameColumn("Area", "Area (" + unit + "^2)");

	}

	setSelectionName(getImageNameForStage(imageIndex, "rgb_input") + " Line Across Gap");

	roiManager("Add");	

	// "(Thick) horizontal line" selection marking the area between the fiducials. Will not correspont to the "rotated rectangle" selection directly, but may allow for better gap threshold finding.

	if (fiducialsAreAlongTheGap) {

		makeLine((xStart + xEnd) / 2 - (xDirectionToLeftSide) * minLeeway * leewayFractionToUse, (yStart + yEnd) / 2,

		         (xStart + xEnd) / 2 + (xDirectionToLeftSide) * minLeeway * leewayFractionToUse, (yStart + yEnd) / 2,

		         distanceFromStartToEnd);

	} else {

		makeLine(xStart, (yStart + yEnd) / 2, xEnd, (yStart + yEnd) / 2, interFiducialSpaceWidth);

	}

	setSelectionName(getImageNameForStage(imageIndex, "rgb_input") + " Horizontal Line Across Gap");

	roiManager("Add");	

}

	run("Duplicate...", "title=[" + getImageNameForStage(imageIndex, "enhanced") + "]");

	// Try to enhance the contrast of the cells while also smoothing out the noise.

	//run("Enhance Contrast...", "saturated=0 normalize");

	//run("Enhance Contrast...", "saturated=5 normalize");

	//run("Enhance Contrast...", "saturated=10 normalize");

	run("Enhance Contrast...", "saturated=20 normalize");  // High values of "saturated" (up to 20 was tested) seem to help with low-contrast images.

	run("Enhance Contrast...", "saturated=" + contrastEnhancementPercentage +" normalize");  // High values of "saturated" (up to 20 was tested) seem to help with low-contrast images.

	run("Median...", "radius=5");  // TODO: Median radius should be smaller than the cell size. (About one third of the SE size used for morphological gradient/opening seems to work.)

}

function determineEmptyRegionThreshold() {

	thresholds = newArray();

	print("\nFinding empty region threshold in the first image, \"" + getImageNameForStage(0, "rgb_input") + "\".");	

	selectWindow(getImageNameForStage(0, "gradient"));

	run("Select None");

	// Find the threshold from all pixels of getImageNameForStage(0, "rgb_input") + " Rectangle Between Fiducials".

	// (Duplicating the rotated-rectangle selection creates a full rectangular image covering the same area.)

	RoiManager.selectByName(getImageNameForStage(0, "rgb_input") + " Rectangle Between Fiducials");	

	run("Duplicate...", "title=[" + getImageNameForStage(0, "temp_for_thresholding") + "]");

	setAutoThreshold(thresholdingMethodToUse);

	getThreshold(lowerRectangleThreshold, upperRectangleThreshold);  // Anything that is <= upperRectangleThreshold will be considered to be the gap.

	print("Threshold calculated using \"" + thresholdingMethodToUse + "\" method from all pixels of \"" + getImageNameForStage(0, "rgb_input") + " Rectangle Between Fiducials\": " + upperRectangleThreshold);

	thresholds = Array.concat(thresholds, upperRectangleThreshold);

	close(getImageNameForStage(0, "temp_for_thresholding"));

	selectWindow(getImageNameForStage(0, "gradient"));

	print("Threshold calculated using \"" + thresholdingMethodToUse + "\" method from all pixels of \"" + getImageNameForStage(0, "rgb_input") + " Rectangle Between Fiducials\": " + upperRectangleThreshold);

	// Find the threshold across the gap (along the "thick line" crossing the gap).

	// Careful: if the line is at an angle other than 90°, the threshold may be too high,

	// because it may happen that none of the columns align with the gap, and so each one's

	// intensity is higher than the intensity of the actual empty background.	

	selectWindow(getImageNameForStage(0, "gradient"));

	RoiManager.selectByName(getImageNameForStage(0, "rgb_input") + " Line Across Gap");	

print("selected image before computation of line profile: \"" + getTitle() + "\"");

print("selected ROI before computation of line profile: \"" + Roi.getName + "\"");

	profile = getProfile();

	// TODO: Maybe the statistics can indicate whether there is any gap present or not? 

	//       But careful -- to test this, use images with a large gap, with no gap at all,

	print("Profile min: " + min + ", max: " + max + ", mean: " + mean + ", stdDev: " + stdDev);

	// Show the profile across the gap.

print("selected image before plotting of line profile: \"" + getTitle() + "\"");

print("selected ROI before plotting of line profile: \"" + Roi.getName + "\"");

	run("Plot Profile");

	rename(intermediateDebugImagePrefix + getTitle());

			setPixel(i, j, profile[i]);

		}

	}

	setAutoThreshold(thresholdingMethodToUse);

	getThreshold(lowerLineThreshold, upperLineThreshold);  // Anything that is <= upperLineThreshold will be considered to be the gap.

	thresholds = Array.concat(thresholds, upperLineThreshold);

	close(getImageNameForStage(0, "temp_for_plot"));

	print("Threshold calculated using \"" + thresholdingMethodToUse + "\" method from plot along \"" + getImageNameForStage(0, "rgb_input") + " Line Across Gap\": " + upperLineThreshold);

	result = Math.min(upperLineThreshold, upperRectangleThreshold);

	print("Returning threshold = min(" + upperLineThreshold + ", " + upperRectangleThreshold + ") = " + result);  // This is only a redundant debugging output.

// TODO: BUG: Sometimes , an error may occur because no linear selection is active when calling run("Plot Profile"). It is unclear when this can happen (seems almost non-deterministic). This has been so far only observed when the horizontal line threshold computation was enabled.

//showMessage("a");

/**/

	// Find the threshold across the gap, perfectly horizontally (along the "thick line" crossing the gap horizontally).

	selectWindow(getImageNameForStage(0, "gradient"));

	RoiManager.selectByName(getImageNameForStage(0, "rgb_input") + " Horizontal Line Across Gap");	

//showMessage("b");

print("selected image before computation of horizontal line profile: \"" + getTitle() + "\"");

print("selected ROI before computation of horizontal line profile: \"" + Roi.getName + "\"");

	profile = getProfile();

	// TODO: Maybe the statistics can indicate whether there is any gap present or not? 

	//       But careful -- to test this, use images with a large gap, with no gap at all,

	//       with a thin gap, and with only a few empty spots left.

	Array.getStatistics(profile, min, max, mean, stdDev);

	print("Profile min: " + min + ", max: " + max + ", mean: " + mean + ", stdDev: " + stdDev);

//showMessage("c");

	// Show the profile across the gap.

print("selected image before plotting of horizontal line profile: \"" + getTitle() + "\"");

print("selected ROI before plotting of horizontal line profile: \"" + Roi.getName + "\"");

	run("Plot Profile");

	rename(intermediateDebugImagePrefix + getTitle() + " (horizontal)");

//showMessage("d");

	// Create a small image containing the plot's values, so that it can be thresholded with one of the built-in thresholding functions.

	plotImageHeight = 1;  // A height greater than 1 wuld be used mostly for debugging/viewing purposes. For most of the thresholding methods, the height of 1 should be enough.

	newImage(getImageNameForStage(0, "temp_for_plot"), bitDepthToImageTypeString(bitDepth()), profile.length, plotImageHeight, 1);

	for (i = 0; i < profile.length; i++) {

		for (j = 0; j < plotImageHeight; j++) {

			setPixel(i, j, profile[i]);

		}

	}

	setAutoThreshold(thresholdingMethodToUse);

	getThreshold(lowerHorizontalLineThreshold, upperHorizontalLineThreshold);  // Anything that is <= upperHorizontalLineThreshold will be considered to be the gap.

	thresholds = Array.concat(thresholds, upperHorizontalLineThreshold);

	close(getImageNameForStage(0, "temp_for_plot"));

	print("Threshold calculated using \"" + thresholdingMethodToUse + "\" method from plot along \"" + getImageNameForStage(0, "rgb_input") + " Horizontal Line Across Gap\": " + upperHorizontalLineThreshold);

/**/

	//result = Math.min(upperLineThreshold, upperRectangleThreshold);

	//print("Returning threshold = min(" + upperLineThreshold + ", " + upperRectangleThreshold + ") = " + result);  // This is only a redundant debugging output.

	//result = Math.min(Math.min(upperLineThreshold, upperRectangleThreshold), upperHorizontalLineThreshold);

	//print("Returning threshold = min(" + upperLineThreshold + ", " + upperRectangleThreshold + ", " + upperHorizontalLineThreshold + ") = " + result);  // This is only a redundant debugging output.

	Array.getStatistics(thresholds, min, max, mean, stdDev);

	result = min;

	print("Candidate thresholds:");  // This is only a redundant debugging output.

	Array.print(thresholds);

	print("Returning min threshold = " + result);

	return result;

}

function processImages() {

	selectIfExists("Log");

	// Check if all images in imagesToAnalyze have the same size, units, and pixel size. If not, abort.

	setBatchMode(true);  // Before testing the image sizes, enter the batch mode in any case to prevent too much flickering. 

	verifyAllImagesHaveIdenticalSizesAndUnits();

var fiducialsAreAlongTheGap;  // True if the fiducials are placed along the gap. False if they are placed to the sides of it.

var gapOrientation;

var contrastEnhancementPercentage;

var closeIntermediateImages;

var useBatchModeForComputations;