/* * Calculate the no of immediate neighbours around a user-defined radius (default: 10 micron) for 2 markers * Ideally, use cell_1 as the reference or pan cell marker, like Hu * and cell_2 as the marker labelling subset of cells * This can be interchanged as long as the names and roi manager files are selected appropriately * There is option to save parametric image */ #@ File (label="Select the maximum projection image") max_proj #@ String (label="Name of celltype 1", description="Cell 1",value="cell_1") cell_type_1 #@ File (label="Select roi manager for cell 1") roi_path_1 #@ String (label="Name of celltype 2", description="Cell 2",value="cell_2") cell_type_2 #@ File (label="Select roi manager for cell 2") roi_path_2 #@ File (style="directory",label="Select Output Folder") save_path #@ File (label="Select roi manager for ganglia (Enter NA if none)",value="NA") roi_ganglia_path #@ String(value="Expand cells by a certain distance so that they touch each other
and then count immediate neighbours (9 micron is default)", visibility="MESSAGE") hint; #@ Double (value=10, min=1, max=15, style="slider",label="Cell expansion distance for cells (microns)") label_dilation #@ boolean save_parametric_image run("Clear Results"); print("\\Clear"); run("Close All"); roiManager("reset"); var fs=File.separator; var fiji_dir=getDirectory("imagej"); var gat_dir=fiji_dir+"scripts"+fs+"GAT"+fs+"Other"+fs+"commands"; //check if label to roi macro is present var label_to_roi=gat_dir+fs+"Convert_Label_to_ROIs.ijm"; if(!File.exists(label_to_roi)) exit("Cannot find label to roi script. Returning: "+label_to_roi); //check if roi to label macro is present var roi_to_label=gat_dir+fs+"Convert_ROI_to_Labels.ijm"; if(!File.exists(roi_to_label)) exit("Cannot find roi to label script. Returning: "+roi_to_label); if(cell_type_1==cell_type_2 || roi_path_1== roi_path_2) exit("Same name or ROI Manager selected"); //open the image open(max_proj); file_name=File.nameWithoutExtension; file_name_length=lengthOf(file_name); if(file_name_length>50) file_name=substring(file_name, 0, 39); //Restricting file name length as in Windows long path names can cause errors getPixelSize(unit, pixelWidth, pixelHeight); if(unit!="microns") print("Image is not in calibrated in microns. Output maybe in pixels"); img=getTitle(); //binary image for ganglia ganglia_binary=""; setOption("BlackBackground", true); if(File.exists(roi_ganglia_path)) { roiManager("open", roi_ganglia_path); //convert ROIs to label map image. It will use dimensions of max projection image above //run("ROI Manager to LabelMap(2D)"); runMacro(roi_to_label); wait(10); run("Select None"); run("Remove Overlay"); //getMinAndMax(min, max); setThreshold(0.5, 65535); run("Convert to Mask"); rename("Ganglia_outline"); ganglia_binary=getTitle(); run("Divide...", "value=255"); //binary image needs to have 0 and 1 as background and foreground, esp to use in clij setMinAndMax(0, 1); //waitForUser; roiManager("reset"); } else ganglia_binary="NA"; roiManager("reset"); roiManager("open", roi_path_1); runMacro(roi_to_label); wait(10); rename(cell_type_1); cell_1=getTitle(); roiManager("show none"); run("Select None"); run("Remove Overlay"); roiManager("reset"); roiManager("open", roi_path_2); runMacro(roi_to_label); wait(10); rename(cell_type_2); cell_2=getTitle(); roiManager("show none"); roiManager("reset"); run("Select None"); run("Remove Overlay"); //label_dilation=9; //9 micron dilation or whatever user enters //convert to pixels label_dilation=round(label_dilation/pixelWidth); label_overlap_1=neighbour_count(cell_1,cell_2,label_dilation,ganglia_binary,cell_type_1,cell_type_2); roiManager("open", roi_path_1); run("Set Measurements...", "min redirect=None decimal=3"); roiManager("Measure"); selectWindow("Results"); cell_count_1=Table.getColumn("Max"); run("Clear Results"); label_overlap_2=neighbour_count(cell_2,cell_1,label_dilation,ganglia_binary,cell_type_2,cell_type_1); roiManager("open", roi_path_2); run("Set Measurements...", "min redirect=None decimal=3"); roiManager("Measure"); selectWindow("Results"); cell_count_2=Table.getColumn("Max"); run("Clear Results"); table_path=save_path+fs+file_name+".csv"; Table.create("Cell counts_overlap"); Table.setColumn("No of "+cell_type_2+" around "+cell_type_1, cell_count_1); Table.setColumn("No of "+cell_type_1+" around "+cell_type_2, cell_count_2); Table.update; Table.save(table_path); if(save_parametric_image==true) { overlap_cell_1=get_parameteric_img(label_overlap_1,cell_1); overlap_cell_2=get_parameteric_img(label_overlap_2,cell_2); selectWindow(overlap_cell_1); saveAs("Tiff", save_path+fs+overlap_cell_1); selectWindow(overlap_cell_2); saveAs("Tiff", save_path+fs+overlap_cell_2); } close("*"); function neighbour_count(ref_img,marker_img,dilate_radius,ganglia_outline,cell_type_1,cell_type_2) { // Init GPU run("CLIJ2 Macro Extensions", "cl_device="); //Ext.CLIJ2_clear(); Ext.CLIJ2_push(ref_img); Ext.CLIJ2_push(marker_img); // // Dilate Labels Ext.CLIJ2_dilateLabels(ref_img, ref_dilate, dilate_radius); // Dilate Labels Ext.CLIJ2_dilateLabels(marker_img, marker_dilate, dilate_radius); if(ganglia_outline!="NA") { Ext.CLIJ2_push(ganglia_outline); Ext.CLIJ2_mask(ref_dilate, ganglia_outline, ref_dil_ganglia); Ext.CLIJ2_mask(marker_dilate, ganglia_outline, marker_dil_ganglia); //Ext.CLIJ2_pull(ref_dil_ganglia); //Ext.CLIJ2_pull(marker_dil_ganglia); //exit("error message"); Ext.CLIJ2_release(ref_dilate); Ext.CLIJ2_release(marker_dilate); //as we are using a mask, it may clip cells and we want to get rid of any incomplete cells . We are using circularity as a way for this //Any cells that are less than 0.4 circularity; get rid of them Ext.CLIJx_morphoLibJCircularityMap(ref_dil_ganglia, ref_circularity); // Exclude Labels With Values Out Of Range minimum_value_range = 0.4; maximum_value_range = 1.0; Ext.CLIJ2_excludeLabelsWithValuesOutOfRange(ref_circularity, ref_dil_ganglia, ref_dilate1, minimum_value_range, maximum_value_range); //image_2 Ext.CLIJx_morphoLibJCircularityMap(marker_dil_ganglia, marker_circularity); Ext.CLIJ2_excludeLabelsWithValuesOutOfRange(marker_circularity, marker_dil_ganglia, marker_dilate1, minimum_value_range, maximum_value_range); Ext.CLIJ2_release(marker_circularity); Ext.CLIJ2_release(ref_circularity); // Label Overlap Count Map Ext.CLIJ2_labelOverlapCountMap(ref_dilate1, marker_dilate1, label_overlap_count); } else Ext.CLIJ2_labelOverlapCountMap(ref_dilate, marker_dilate, label_overlap_count); Ext.CLIJ2_pull(ref_img); Ext.CLIJ2_pull(marker_img); Ext.CLIJ2_pull(label_overlap_count); rename("No of "+cell_type_2+" around "+cell_type_1); label_overlap_count=getTitle(); run("Fire"); return label_overlap_count; } function get_parameteric_img(label_overlap_img,cell_img) { run("CLIJ2 Macro Extensions", "cl_device="); Ext.CLIJ2_push(label_overlap_img); Ext.CLIJ2_push(cell_img); // covert label overlap img to binary constant = 1.0; Ext.CLIJ2_greaterOrEqualConstant(cell_img, label_bin, constant); // Multiply Images Ext.CLIJ2_multiplyImages(label_overlap_img, label_bin, parametric_img); Ext.CLIJ2_release(label_bin); Ext.CLIJ2_release(label_overlap_img); if(isOpen(label_overlap_img)) close(label_overlap_img); Ext.CLIJ2_pull(parametric_img); selectWindow(parametric_img); rename(label_overlap_img); parametric_img=getTitle(); return parametric_img; }