From b4aa8c6ed8e9b9b05f4e71c93f5f8a4acc57d810 Mon Sep 17 00:00:00 2001
From: glynnelson <glyn.nelson@ncl.ac.uk>
Date: Wed, 8 Jul 2020 17:20:56 +0100
Subject: [PATCH] Add files via upload

---
 ...i_counting_wholedir_20x_moments_thresh.txt |  81 +++++++
 Foci_counting_wholedir_Choose_thresh.ijm      | 223 ++++++++++++++++++
 George_mitophagy_Dmi8_looped.ijm              | 130 ++++++++++
 ROI_overlay_measure_macro.ijm                 |  39 +++
 Split 2 channels Zstacks_v1.0.ijm             |  67 ++++++
 Yoana_Shrink_ROIv3.ijm                        | 166 +++++++++++++
 convert_lif_files_3.ijm                       |  95 ++++++++
 convert_to_ome_tiff_with choose_filetype.ijm  | 101 ++++++++
 convert_to_tiff_with choose_filetype.ijm      | 101 ++++++++
 extract_qptiff_IMAGES_whole_dir.ijm           |  51 ++++
 flatten_CZI_IMAGES_whole_dir.txt              |  38 +++
 flatten_ZVI_IMAGES_whole_dir.txt              |  38 +++
 ...c_counting_wholedir_20x_moments_thresh.txt | 107 +++++++++
 ...ample_counting_WholeDir_manualSampling.ijm | 130 ++++++++++
 manual_spot_coutning_WholeDir_example.ijm     |  90 +++++++
 measure_multiImage_CZI_5chMP.ijm              | 135 +++++++++++
 per_nuc_FISH_modified_Tengfei.txt             |  95 ++++++++
 realign_20x_DM5500_Cy5cube.ijm                |  82 +++++++
 18 files changed, 1769 insertions(+)
 create mode 100644 Eva_foci_counting_wholedir_20x_moments_thresh.txt
 create mode 100644 Foci_counting_wholedir_Choose_thresh.ijm
 create mode 100644 George_mitophagy_Dmi8_looped.ijm
 create mode 100644 ROI_overlay_measure_macro.ijm
 create mode 100644 Split 2 channels Zstacks_v1.0.ijm
 create mode 100644 Yoana_Shrink_ROIv3.ijm
 create mode 100644 convert_lif_files_3.ijm
 create mode 100644 convert_to_ome_tiff_with choose_filetype.ijm
 create mode 100644 convert_to_tiff_with choose_filetype.ijm
 create mode 100644 extract_qptiff_IMAGES_whole_dir.ijm
 create mode 100644 flatten_CZI_IMAGES_whole_dir.txt
 create mode 100644 flatten_ZVI_IMAGES_whole_dir.txt
 create mode 100644 foci_and_coloc_counting_wholedir_20x_moments_thresh.txt
 create mode 100644 manual_randomSample_counting_WholeDir_manualSampling.ijm
 create mode 100644 manual_spot_coutning_WholeDir_example.ijm
 create mode 100644 measure_multiImage_CZI_5chMP.ijm
 create mode 100644 per_nuc_FISH_modified_Tengfei.txt
 create mode 100644 realign_20x_DM5500_Cy5cube.ijm

diff --git a/Eva_foci_counting_wholedir_20x_moments_thresh.txt b/Eva_foci_counting_wholedir_20x_moments_thresh.txt
new file mode 100644
index 0000000..3199b1f
--- /dev/null
+++ b/Eva_foci_counting_wholedir_20x_moments_thresh.txt
@@ -0,0 +1,81 @@
+//Assumes the image is a tif with DAPI in channel1, H2AX in channel2 
+//  It looks for nuclei then counts respective spots for each nucleus.
+//All results get stored in a concatenating results file.
+//Plus the mask images are stored (all 2 original images converted to masks)
+// so you can see what it did.
+// altered so find maxima works on raw data,not thresholded.
+// var tolerance = 30; removed this and set manually in find maxima.
+run("Set Measurements...", "area integrated limit display redirect=None decimal=0");
+
+dir1 = getDirectory("Choose folder with tif files ");
+list = getFileList(dir1);
+
+setBatchMode(true);
+
+// create folders for the tifs
+	dir1parent = File.getParent(dir1);
+	dir1name = File.getName(dir1);
+	dir2 = dir1parent+File.separator+dir1name+"_masks";
+	if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for mask images
+		}
+
+for (i=0; i<list.length; i++) {
+	showProgress(i+1, list.length);
+	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+	filename = dir1 + list[i];
+	if (endsWith(filename, "tif")) {
+		path=dir1+list[i];
+		run("Bio-Formats Importer", "open=["+filename+"] autoscale color_mode=Default view=Hyperstack stack_order=XYCZT"); 
+		originalImage = getTitle();
+		roiManager("Reset");
+		selectWindow(originalImage);
+		run("Duplicate...", "title=nuc duplicate channels=2"); //Change this to the DAPI channel slice*******
+		wait(1000);
+		setAutoThreshold("Moments dark");
+		run("Threshold");
+		run("Watershed");
+		run("Analyze Particles...", "size=48-2500 circularity=0.04-1.00 exclude add");
+		run("Blue");
+		run("16-bit");
+//nuclei now added to ROI manager
+
+//next extract h2ax channel
+		selectWindow(originalImage);
+		run("Duplicate...", "title=h2ax duplicate channels=1");
+		wait(1000);
+
+//next find H2AX foci per nuc
+		selectWindow("h2ax");
+		rename(originalImage+"_h2ax");
+		for(j=0; j<roiManager("count"); j++) {
+			roiManager("select", j);
+			run("Find Maxima...", "noise=100 output=[Count]");
+//	run("Find Maxima...", "noise="+tolerance+" output=[Point Selection]");
+			run("Add Selection...");
+			}
+		saveAs("Results", dir2+File.separator+originalImage+"_h2ax.xls"); //This is saving as it goes along in case it crashes
+		selectWindow(originalImage+"_h2ax");
+		rename("h2ax2");
+		run("Enhance Contrast", "saturated=0.35");
+		selectWindow(originalImage);
+		wait(1000);
+		close();
+
+		run("Merge Channels...", "c1=h2ax2 c2=nuc create");
+		rename("masks");
+
+		saveAs("TIFF", dir2+File.separator+originalImage+"_ThresholdMasks.tif");
+
+
+	close();
+	}
+setBatchMode(false);
+}
+
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");
+
+// macro 
+
+
+
diff --git a/Foci_counting_wholedir_Choose_thresh.ijm b/Foci_counting_wholedir_Choose_thresh.ijm
new file mode 100644
index 0000000..a571a09
--- /dev/null
+++ b/Foci_counting_wholedir_Choose_thresh.ijm
@@ -0,0 +1,223 @@
+/* Macro to count spots inside other spots
+ *  Updated March 2020
+ *  It now asks whichc hannels the nuclear counterstain and the spots are in (can work with more than 2 channel images)
+ *  It still assumes the image is a tif
+ *  It looks for nuclei then counts respective spots for each nucleus.
+ *  All results get stored in a concatenating results file.
+ *  Plus the mask images are stored (all 2 original images converted to masks)
+ *  so you can see what it did.
+ *  
+ *  Input:
+ *  Assumes the image is a tif (any number of channels but it has to be 1 z, not a stack) 
+ *  It asks for the channel numbers for nuclear counterstain and sub nculear spots
+ *  
+ *  Note- tifs can be produced via another macro to convert from proprietary file format
+ *  
+ *  Then you are asked to review the threshold stack it has created and choose the best method.
+ *  NOTE- It does NOT work if you choose Huang2!!!!  Some weird choice of 2 different lists in ImageJ.
+ *  
+ *  Output:
+ *  All results get stored in a concatenating results file.
+ *  Plus the mask images are stored (all 2 original images converted to masks) so you can check on accuracy.
+ *  
+ *  To do: convert to python- way easier than this clunky thing, and would be able to deal with 3D datasets.
+ *  Add to run from bioformats and only use the chosen filetype (easy enough)
+ *  
+ *  
+ *  Glyn Nelson, Newcastle University Bioimaging Unit
+ */
+
+//Variables need to be defined if used between functions and the main macro, not just for sharing between macros.
+var fred;
+var FociChannel =1;
+var DapiChannel = 2;
+run("Set Measurements...", "area integrated limit display add redirect=None decimal=0");
+
+setChannelNames();
+
+dir1 = getDirectory("Choose folder with tif files ");
+list = getFileList(dir1);
+
+setBatchMode(true);
+
+// create folders for the tifs
+	dir1parent = File.getParent(dir1);
+	dir1name = File.getName(dir1);
+	dir2 = dir1parent+File.separator+dir1name+"_masks";
+	if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for mask images
+		}
+for (i=0; i<list.length; i++) {
+	showProgress(i+1, list.length);
+	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+	filename = dir1 + list[i];
+	if (endsWith(filename, "tif")) {
+		path=dir1+list[i];
+		run("Bio-Formats Importer", "open=["+filename+"] autoscale color_mode=Default view=Hyperstack stack_order=XYCZT"); 
+		originalImage = getTitle();
+		roiManager("Reset");
+		selectWindow(originalImage);
+		run("Make Composite", "display=Composite");
+//		Stack.getDimensions(width, height, channels, slices, frames);
+//		run("Stack to Hyperstack...", "order=xyczt(default) channels=channels slices=slices frames=frames display=Color");
+		run("Duplicate...", "title=nuc duplicate channels=" +DapiChannel);
+		selectWindow("nuc");
+		run("Grays");
+		BuildThreshStack();  //I am absolutely damned if I can get this to work.  it insists that it the variable ThresholdChoice doesn't exist once I go through this as a function.
+		selectWindow("nuc");
+		setOption("BlackBackground", true);
+		setAutoThreshold(fred);
+		run("Threshold");
+		run("Watershed");
+		run("Analyze Particles...", "size=48-2500 circularity=0.04-1.00 exclude add");
+		run("Blue");
+		run("16-bit");
+//nuclei now added to ROI manager
+
+//next extract h2ax channel
+		selectWindow(originalImage);
+		run("Duplicate...", "title=h2ax duplicate channels=FociChannel");
+		run("16-bit");
+		wait(1000);
+
+//next find H2AX foci per nuc
+		selectWindow("h2ax");
+		rename(originalImage+"_h2ax");
+		for(j=0; j<roiManager("count"); j++) {
+			roiManager("select", j);
+			run("Find Maxima...", "noise=100 output=[Count]");
+//	run("Find Maxima...", "noise="+tolerance+" output=[Point Selection]");
+			run("Add Selection...");
+			}
+		saveAs("Results", dir2+File.separator+originalImage+"_h2ax.xls"); //This is saving as it goes along in case it crashes
+		selectWindow(originalImage+"_h2ax");
+		rename("h2ax2");
+		run("Enhance Contrast", "saturated=0.35");
+		selectWindow(originalImage);
+		wait(1000);
+		close();
+		run("Merge Channels...", "c1=h2ax2 c2=nuc create");
+		rename("masks");
+		saveAs("TIFF", dir2+File.separator+originalImage+"_ThresholdMasks.tif");
+	close();
+	}
+setBatchMode(false);
+}
+
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");
+
+// macro 
+
+
+//FUNCTION- determine channel order
+function setChannelNames() {
+Dialog.create("Set which channels are which");  //enable use interactivity
+Dialog.addMessage("Which channel is DAPI/ nuclear counterstain in?");
+Dialog.addString(" DAPI Channel", DapiChannel);	//add extension
+Dialog.addMessage("Which channel are the sub-nuclear spots labelled in?");
+Dialog.addString("Foci Channel", FociChannel);	//add extension
+Dialog.addMessage("Use channel numbers (eg 1,2) to set the channels to use for determining the nuclei and spots");
+Dialog.show();
+//StringDapiChannel = Dialog.getString(DapiChannel);  ******Apparently don't need to convert a number from a string- it figures it out itself.
+//Dapi = parseFloat(StringDapiChannel);
+//StringFociChannel = Dialog.getString(FociChannel);
+//Foci = parseFloat(StringFociChannel);
+}
+//need to set var Foci and var Dapi
+//End of function setChannelNames
+
+
+
+
+//FUNCTION Choose global Threshold Method
+function BuildThreshStack() {
+	run("Auto Threshold", "method=[Try all] white show");
+selectWindow("Montage"); 
+run("Montage to Stack...", "images_per_row=5 images_per_column=4 border=0");
+run("RGB Color");
+run("Colors...", "foreground=green background=black selection=red");
+ style = 4; // Font.BOLD
+ call("ij.gui.TextRoi.setFont", "Sans", 32, style) 
+
+//This is nuts, but I've spent so long trying to use arrays or lists to run through this labelling that I've given up.  
+//Writting the next 34 lines took less than 10 minutes. Grrrrrrrrr
+//run("Label...", "format=0000 x=14 y=20 font=32 text=Method use use_text"); 
+setSlice(1);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Default] range=1-1");
+setSlice(2);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Huang] range=2-2");
+setSlice(3);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Huang2] range=3-3");
+setSlice(4);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Intermodes] range=4-4");
+setSlice(5);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[IsoData] range=5-5");
+setSlice(6);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[li] range=6-6");
+setSlice(7);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[MaxEntropy] range=7-7");
+setSlice(8);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Mean] range=8-8");
+setSlice(9);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[MinError] range=9-9");
+setSlice(10);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Minimum] range=10-10");
+setSlice(11);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Moments] range=11-11");
+setSlice(12);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Otsu] range=12-12");
+setSlice(13);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Percentile] range=13-13");
+setSlice(14);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[RenyiEntropy] range=14-14");
+setSlice(15);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Shahbhag] range=15-15");
+setSlice(16);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Triangle] range=16-16");
+setSlice(17);
+run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Yen] range=17-17");
+
+
+waitForUser("Browse the Stack to choose the best Method \n then click OK to input your choice");
+Dialog.create("Choose your desired threshold");  //enable use interactivity
+ Dialog.addChoice("Best threshold is:", newArray("Default", "Huang","Huang2","Intermodes","IsoData","Li","MaxEntropy","Mean","MinError","Minimum","Moments","Otsu","Percentile","RenyiEntropy","Shanbhag","Triangle","Yen"));
+Dialog.show();
+   fred = Dialog.getChoice();
+
+selectWindow("Stack");
+close();
+
+selectWindow("Montage");
+close();
+}
+
+//ensure var fred is set
+//End of function BuildThreshStack
+
+
+//Neither of the functions belwo are in use in this version.
+
+//FUNCTION- choose autoThreshold method
+function choose_me() {
+	waitForUser("Browse the Stack to choose the best Method \n then click OK to input your choice");
+Dialog.create("Choose your desired threshold");  //enable use interactivity
+ Dialog.addChoice("Best threshold is:", newArray("Default", "Huang","Huang2","Intermodes","IsoData","Li","MaxEntropy","Mean","MinError","Minimum","Moments","Otsu","Percentile","RenyiEntropy","Shanbhag","Triangle","Yen"));
+Dialog.show();
+   fred = Dialog.getChoice();
+
+//print("diaolog gives fred is ", fred);
+
+}
+//need to set var fred
+//End of function choose_me
+
+
+//FUNCTION Call_Choice
+function Call_Choice(){
+	waitForUser("Browse the Stack to choose the best Method \n number then click OK to input your choice");
+	Desired_Threshold_Method = getNumber("Choose your Preferred Threshold Number", 0);
+	//Stack.getPosition(slice);
+	//print ("Your Choice is...", Desired_Threshold_Method);
+}
+//ensure global var Desired_Threshold_Method is set
+//End of Function Call_Choices
\ No newline at end of file
diff --git a/George_mitophagy_Dmi8_looped.ijm b/George_mitophagy_Dmi8_looped.ijm
new file mode 100644
index 0000000..c6b8266
--- /dev/null
+++ b/George_mitophagy_Dmi8_looped.ijm
@@ -0,0 +1,130 @@
+macro 'mitophagy analysis in LIFs' {
+
+/*
+ * - Based on earlier macro that converts LIF files into TIFF. 
+ * - the macro ask for an input folder that should contain 1 or more lif files. Nothing else.
+ * - each series is saved as one tiff. The name of the series is appended to the file name.
+ * - if the series has more than one frame, a Max Intensity projection is also performed 
+ * - before saving. The TIFFs are saved in a folder generated by the macro in the same 
+ * - parent folder as the lifs, with the same prefix for the folder name, with TIFF suffix.
+ * 
+ * Martin Hoehne, August 2015
+ * 
+ *  Update October 2015: works for Multichannel images as well
+ *
+ * Update Jan2016 (Glyn Nelson).  Now also saves z stack as well as mip if it is a z stack.
+ *  This works with SP8 images as long as they aren't in subfolders, such as Mark and Find: these
+ *  images have a '/' in their name, so it throws an error when trying to save. 
+ * Update Jan2016 (Glyn Nelson).  Fixed.  It will now remove '/' if it exists in the imagename before saving. 
+ * 
+ * Update Feb 2016 (Glyn Nelson).  Now ignores other files in the folder with lifs (although it will give multiple finished
+ *  messages for each extra file it finds...)
+ *
+ * 
+ * 
+ * September2019.  Modified to automatically remove green signal from red mitphogay data in George's Dmi8 images
+ * REQUIREMENTS: 
+ * mito-mKeima transfected cells used for this- ratiometric, excitation dependent upon pH
+ *  https://www.nature.com/articles/nprot.2017.060 
+ *  https://www.fpbase.org/protein/mkeima/
+ * Single plane lif files with 2 channels, green (440ex) in channel1 and red (560ex) in channel2
+ * (can have more channels, but these two need to be the first and in this order)
+ * 
+ * Notes.  Could add to work on z stacks too, either as a projection, or each slice one at a time (would worry
+ * about spurious results from the out of focus planes in thsi case though).
+ */
+
+run("Bio-Formats Macro Extensions");
+
+
+dir1 = getDirectory("Choose folder with lif files ");
+list = getFileList(dir1);
+
+setBatchMode(true);
+
+// create folders for the tifs
+	dir1parent = File.getParent(dir1);
+	dir1name = File.getName(dir1);
+	dir2 = dir1parent+File.separator+dir1name+"_Tiffs";
+	if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for tiff
+		}
+ 
+for (i=0; i<list.length; i++) {
+	showProgress(i+1, list.length);
+	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+	filename = dir1 + list[i];
+		if (endsWith(filename, "lif")){
+		path=dir1+list[i];
+
+	//how many series in this lif file?
+		Ext.setId(path);//-- Initializes the given path (filename).
+		Ext.getSeriesCount(seriesCount); //-- Gets the number of image series in the active dataset.
+	
+		for (j=1; j<=seriesCount; j++) {
+		
+		run("Bio-Formats", "open=path autoscale color_mode=Default view=Hyperstack stack_order=XYCZT series_"+j);
+	
+		name=File.nameWithoutExtension;
+
+	//retrieve name of the series from metadata
+		text=getMetadata("Info");
+		n1=indexOf(text," Name = ")+8;// the Line in the Metadata reads "Series 0 Name = ". Complete line cannot be taken, because
+									  // The number changes of course. But at least in the current version of Metadata this line is the 
+									  // only occurence of " Name ="
+		n2=indexOf(text,"SizeC = ");  // this is the next line in the Metadata
+		extractedname=substring(text,n1,n2-2);
+		cleanname=split(extractedname,"/");  // this is done here to remove the forward slash that is found in the image name 
+					// if it is part of a mark and find subfolder, for example 
+
+//We still want both parts of the filenename.  So if there are two parts to the array, then stick them together, otherwise just take the first:
+			if (cleanname.length==2){
+			seriesname=cleanname[0]+cleanname[1];
+			}		
+			else seriesname=cleanname[0];
+	//Make masked Red channel with green removed and low signal red removed		
+			
+			rename("orig");
+			run("Duplicate...", "title=green duplicate channels=1");
+			setAutoThreshold("MaxEntropy dark");
+			setOption("BlackBackground", false);
+			run("Convert to Mask");
+			run("16-bit");
+			run("Multiply...", "value=257.000");
+
+			selectWindow("orig");
+			run("Duplicate...", "title=red duplicate channels=2");
+
+			imageCalculator("Subtract create", "red","green");
+			selectWindow("Result of red");
+			rename("redMasked");
+
+			selectWindow("redMasked");
+			setAutoThreshold("MaxEntropy dark");
+			run("Convert to Mask");
+			run("16-bit");
+			run("Multiply...", "value=257.000");
+			selectWindow("redMasked");
+			run("Invert");
+
+			imageCalculator("Subtract create", "red","redMasked");
+			selectWindow("Result of red");
+			rename("redFinal");
+	//stick modified red image onto end of orignal image and save as stack
+			run("Concatenate ", "  title=[Concat] image_1=[orig] image_2=[redFinal] image_3=[-- None --]");
+	//		Stack.setDisplayMode("composite");
+			run("Make Composite", "display=Composite");
+			saveAs("TIFF", dir2+File.separator+name+"_"+seriesname+".tif");
+
+			run("Close All");
+//		run("Collect Garbage");
+		
+	}
+
+}
+run("Close All");
+setBatchMode(false);
+
+}
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
+ // macro
diff --git a/ROI_overlay_measure_macro.ijm b/ROI_overlay_measure_macro.ijm
new file mode 100644
index 0000000..9c6444c
--- /dev/null
+++ b/ROI_overlay_measure_macro.ijm
@@ -0,0 +1,39 @@
+
+macro "measure and label" {
+
+if (nImages==0) {
+	exit("No images are open");
+}
+
+imageTitle = getTitle();
+type = selectionType();
+
+// in case the user forgot to make a selection
+if (type == -1) {
+	waitForUser("Draw a ROI on the image " + imageTitle + " and press OK");
+}
+
+type = selectionType();
+
+// check selection type and measure to perform
+if (type==1 || type==2) {
+    field = "Area";
+} else if (type==5) {
+    field = "Length";
+} else if (type==8) {
+    field = "Angle";
+} else {
+    exit("I don't know what to do");
+}
+
+run("Measure");
+label = "" + getResult(field, nResults-1);
+
+// get top left coordinates of bounding box
+getSelectionBounds(x, y, width, height);
+
+// add selection and measure as overlays
+Overlay.addSelection;
+Overlay.drawString(label, x, y)
+
+}
diff --git a/Split 2 channels Zstacks_v1.0.ijm b/Split 2 channels Zstacks_v1.0.ijm
new file mode 100644
index 0000000..ec25905
--- /dev/null
+++ b/Split 2 channels Zstacks_v1.0.ijm	
@@ -0,0 +1,67 @@
+// -------------------------------------------------------------------
+// Written by: Patrice Mascalchi
+// Date: 2020-02
+// DRVision Technologies LLC
+// -------------------------------------------------------------------
+
+// **** manual parameters ****
+nCh = 2;
+
+// ********************************
+
+// Get image directory ---------------
+path = getDirectory("Choose the directory containing your files");
+flist = SortFileList(path, ".tif");
+if (!endsWith(path, File.separator)) path = path + File.separator;
+
+// Get name of selected folder
+tmp = File.getParent(path);
+parentDir = substring(path, lengthOf(tmp)+1);
+if (!endsWith(parentDir, File.separator)) parentDir = parentDir + File.separator;
+if (lengthOf(parentDir) > 20) parentDir = substring(parentDir, 0, 20);
+
+// Defining output directory
+outdirs = newArray(nCh);
+for (o=0; o<nCh; o++) {
+    outdirs[o] = path + "C" + o+1 + "-" + parentDir;
+    if (!File.exists(outdirs[o])) File.makeDirectory(outdirs[o]);
+}
+
+setBatchMode(true);
+
+// Processing the files
+for (f=0; f<flist.length; f++) {
+    fname = flist[f];
+    // run("Bio-Formats Importer", "open=[" + path + flist[f] +"] autoscale color_mode=Composite view=Hyperstack stack_order=Default);
+    open(path + fname);
+    print("Processing "+ f+1 +"/"+ flist.length +": "+ fname);
+
+    nSl = nSlices / nCh;
+
+    run("Stack to Hyperstack...", "order=xyczt(default) channels="+nCh+" slices="+nSl+" frames=1 display=Color");
+
+    run("Split Channels");
+    
+    for (c=1; c<=nCh; c++) {
+        selectWindow("C"+ c +"-"+ fname); 
+        saveAs(outdirs[c-1] +"C"+ c +"-"+ fname);
+    }
+    run("Close All");
+}
+setBatchMode(false);
+print("Done!");
+
+// --------------------------------------------------
+function SortFileList(path, filter) {		// Sort with endsWith
+	flist = getFileList(path);
+	OutAr = newArray(flist.length);
+	ind = 0;
+	for (f=0; f<flist.length; f++) {
+		//print(flist[f] + " : "+endsWith(flist[f], filter));
+		if (endsWith(flist[f], filter)) {
+			OutAr[ind] = flist[f];
+			ind++;
+		}
+	}
+	return Array.trim(OutAr, ind);
+}
diff --git a/Yoana_Shrink_ROIv3.ijm b/Yoana_Shrink_ROIv3.ijm
new file mode 100644
index 0000000..b0fc827
--- /dev/null
+++ b/Yoana_Shrink_ROIv3.ijm
@@ -0,0 +1,166 @@
+// Wrote this to duplicate work done in Starling et al EMBO Reports 2016 paper:
+// ‘Folliculin directs the formation of a Rab34–RILP complex to control the nutrient‐dependent dynamic distribution of lysosome’
+// It creates ROIs that shrink by 10 % in size each time, and then reports the results for each ROI.
+// I've used the minor axis from shape descriptors- I tried Ferets, but it didn't work very well with odd shapes.
+// Secondly, it creates a new image of the first 10% ring (the near-membrane portion) for downstream analysis.
+// It doesn't save the reuslts currently.
+// It requires an image with an ROI drawn in it.
+// It saves the ring tif image in the same directory that the original image was opened from (haven't tested with lifs etc though!)
+
+// Glyn Nelson, Bioimaging Unit, Newcastle University.  July 2019.
+
+//Requirements: An open image with an ROI drawn in it.  
+//  If it is a hyperstack, then ensure the channel you wish to analyse is selected.
+
+dir = getDirectory("image");
+name=File.nameWithoutExtension
+run("ROI Manager...");
+nROIs = roiManager("count");
+if (nROIs >0){
+	roiManager("Delete");
+	}
+roiManager("Add");
+roiManager("Select", 0);
+roiManager("Rename", "Outside");
+run("Set Measurements...", "area mean min fit shape feret's integrated display redirect=None decimal=3");
+run("Measure");
+Ferets = getResult("Minor");
+//Ferets = getResult("Feret");
+ShrinkFactor = (Ferets*0.1);
+//print (ShrinkFactor);
+for (i = 1; i < 10; i++){
+	Shrink = (Ferets-(i*ShrinkFactor));
+//	getResult("Frame", i) + 1;
+//	Ferets = getResult("Feret", i);
+//	Min = getResult("Min", i);
+//	print(frame + ", " + Ferets + ", " + Min);
+	Scale = (Shrink/Ferets);
+	run("Scale... ", "x=Scale y=Scale centered");
+	roiManager("Add");
+	roiManager("Select", i);
+	roiManager("Rename", i);
+//	print(Ferets + ", " + ShrinkFactor + ", " + Shrink);
+}
+roiManager("Deselect");
+// Need to reset scaling back to original image here  **UPDATE-NO NEED- IT WORKS FINE REGARDLESS OF IMAGE SCALING
+run("Set Measurements...", "area mean integrated display redirect=None decimal=3");
+roiManager("multi-measure measure_all");
+saveAs("Results", dir+File.separator+name+"Decile_Areas100to10pct.csv");
+run("Duplicate...", "title=ForColoc duplicate");
+//rename("ForColoc");
+selectWindow("ForColoc");
+roiManager("Select", 2);
+setBackgroundColor(0, 0, 0);
+run("Clear");
+//run("Clear", "slice");
+roiManager("Select", 1);
+run("Clear Outside");
+saveAs("TIFF", dir +File.separator+name+"MembraneRing10pct.tif");
+
+//Now also get ring values
+rename("10pctRing");
+
+selectWindow(name);
+run("Duplicate...", "title=100pctRing duplicate");
+selectWindow("100pctRing");
+roiManager("Select", 9);
+roiManager("Rename", "100");
+run ("Clear Outside");
+roiManager("multi-measure measure_all");
+close ("100pctRing");
+
+selectWindow("name);
+run("Duplicate...", "title=90pctRing duplicate");
+selectWindow("90pctRing");
+roiManager("Select", 100);
+run("Clear");
+roiManager("Select", 8);
+roiManager("Rename", "90");
+run ("Clear Outside");
+roiManager("multi-measure measure_all append");
+close ("90pctRing");
+
+selectWindow("name);
+run("Duplicate...", "title=80pctRing duplicate");
+selectWindow("80pctRing");
+roiManager("Select", 90);
+run("Clear");
+roiManager("Select", 7);
+roiManager("Rename", "80");
+run ("Clear Outside");
+roiManager("multi-measure measure_all append");
+close ("80pctRing");
+
+selectWindow("name);
+run("Duplicate...", "title=70pctRing duplicate");
+selectWindow("70pctRing");
+roiManager("Select", 80);
+run("Clear");
+roiManager("Select", 6);
+roiManager("Rename", "70");
+run ("Clear Outside");
+roiManager("multi-measure measure_all append");
+close ("70pctRing");
+
+selectWindow("name);
+run("Duplicate...", "title=60pctRing duplicate");
+selectWindow("60pctRing");
+roiManager("Select", 70);
+run("Clear");
+roiManager("Select", 5);
+roiManager("Rename", "60");
+run ("Clear Outside");
+roiManager("multi-measure measure_all append");
+close ("60pctRing");
+
+selectWindow("name);
+run("Duplicate...", "title=50pctRing duplicate");
+selectWindow("50pctRing");
+roiManager("Select", 60);
+run("Clear");
+roiManager("Select", 4);
+roiManager("Rename", "50");
+run ("Clear Outside");
+roiManager("multi-measure measure_all append");
+close ("50pctRing");
+
+selectWindow("name);
+run("Duplicate...", "title=40pctRing duplicate");
+selectWindow("40pctRing");
+roiManager("Select", 50);
+run("Clear");
+roiManager("Select", 3);
+roiManager("Rename", "40");
+run ("Clear Outside");
+roiManager("multi-measure measure_all append");
+close ("40pctRing");
+
+selectWindow("name);
+run("Duplicate...", "title=30pctRing duplicate");
+selectWindow("30pctRing");
+roiManager("Select", 40);
+run("Clear");
+roiManager("Select", 2);
+roiManager("Rename", "30");
+run ("Clear Outside");
+roiManager("multi-measure measure_all append");
+close ("30pctRing");
+
+selectWindow("name);
+run("Duplicate...", "title=20pctRing duplicate");
+selectWindow("20pctRing");
+roiManager("Select", 30);
+run("Clear");
+roiManager("Select", 1);
+roiManager("Rename", "20");
+run ("Clear Outside");
+roiManager("multi-measure measure_all append");
+close ("20pctRing");
+
+selectWindow("10pctRing");
+roiManager("Select", Outside);
+roiManager("Rename", "10");
+roiManager("multi-measure measure_all append");
+close ("10pctRing");
+
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
diff --git a/convert_lif_files_3.ijm b/convert_lif_files_3.ijm
new file mode 100644
index 0000000..051cb36
--- /dev/null
+++ b/convert_lif_files_3.ijm
@@ -0,0 +1,95 @@
+macro 'convert LIF tiff' {
+
+/*
+ * - converts LIF files into TIFF. 
+ * - the macro ask for an input folder that should contain 1 or more lif files. Nothing else.
+ * - each series is saved as one tiff. The name of the series is appended to the file name.
+ * - if the series has more than one frame, a Max Intensity projection is also performed 
+ * - before saving. The TIFFs are saved in a folder generated by the macro in the same 
+ * - parent folder as the lifs, with the same prefix for the folder name, with TIFF suffix.
+ * 
+ * Martin Hoehne, August 2015
+ * 
+ *  Update October 2015: works for Multichannel images as well
+ *
+ * Update Jan2016 (Glyn Nelson).  Now also saves z stack as well as mip if it is a z stack.
+ *  This works with SP8 images as long as they aren't in subfolders, such as Mark and Find: these
+ *  images have a '/' in their name, so it throws an error when trying to save. 
+ * Update Jan2016 (Glyn Nelson).  Fixed.  It will now remove '/' if it exists in the imagename before saving. 
+ * 
+ * Update Feb 2016 (Glyn Nelson).  Now ignores other files in the folder with lifs (although it will give multiple finished
+ *  messages for each extra file it finds...)
+ */
+
+run("Bio-Formats Macro Extensions");
+
+
+dir1 = getDirectory("Choose folder with lif files ");
+list = getFileList(dir1);
+
+setBatchMode(true);
+
+// create folders for the tifs
+	dir1parent = File.getParent(dir1);
+	dir1name = File.getName(dir1);
+	dir2 = dir1parent+File.separator+dir1name+"_Tiffs";
+	if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for tiff
+		}
+ 
+for (i=0; i<list.length; i++) {
+	showProgress(i+1, list.length);
+	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+	filename = dir1 + list[i];
+		if (endsWith(filename, "lif")){
+		path=dir1+list[i];
+
+	//how many series in this lif file?
+		Ext.setId(path);//-- Initializes the given path (filename).
+		Ext.getSeriesCount(seriesCount); //-- Gets the number of image series in the active dataset.
+	
+		for (j=1; j<=seriesCount; j++) {
+		
+		run("Bio-Formats", "open=path autoscale color_mode=Default view=Hyperstack stack_order=XYCZT series_"+j);
+	
+		name=File.nameWithoutExtension;
+
+	//retrieve name of the series from metadata
+		text=getMetadata("Info");
+		n1=indexOf(text," Name = ")+8;// the Line in the Metadata reads "Series 0 Name = ". Complete line cannot be taken, because
+									  // The number changes of course. But at least in the current version of Metadata this line is the 
+									  // only occurence of " Name ="
+		n2=indexOf(text,"SizeC = ");  // this is the next line in the Metadata
+		extractedname=substring(text,n1,n2-2);
+		cleanname=split(extractedname,"/");  // this is done here to remove the forward slash that is found in the image name 
+					// if it is part of a mark and find subfolder, for example 
+
+//We still want both parts of the filenename.  So if there are two parts to the array, then stick them together, otherwise just take the first:
+			if (cleanname.length==2){
+			seriesname=cleanname[0]+cleanname[1];
+			}		
+			else seriesname=cleanname[0];
+	//project and save
+				if (nSlices>1) {
+				run("Z Project...", "projection=[Max Intensity]");
+				if (nSlices>1) Stack.setDisplayMode("composite");
+				saveAs("Tiff", dir2+File.separator+name+"_"+seriesname+"_MIP.tif");
+				close();
+				Stack.setDisplayMode("composite");
+				saveAs("TIFF", dir2+File.separator+name+"_"+seriesname+".tif");
+			}	
+			else Stack.setDisplayMode("composite");
+			saveAs("TIFF", dir2+File.separator+name+"_"+seriesname+".tif");
+			run("Close All");
+//		run("Collect Garbage");
+		
+	}
+
+}
+//showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
+run("Close All");
+setBatchMode(false);
+
+}
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
+ // macro
diff --git a/convert_to_ome_tiff_with choose_filetype.ijm b/convert_to_ome_tiff_with choose_filetype.ijm
new file mode 100644
index 0000000..e961ef7
--- /dev/null
+++ b/convert_to_ome_tiff_with choose_filetype.ijm	
@@ -0,0 +1,101 @@
+//FUNCTION: opens a dialog to set text list for filtering a list
+//example: setDialogImageFileFilter();
+//this function set interactively the global variables used by the function getFilteredFileList
+//it can be set to ask for other text to filter files on.  I didn't get this to work though and have removed them.
+//this function needs global variables! (see below)
+/*
+
+var fileExtension = ".tif";   												//default definition of extension
+var filterStrings = newArray("","","");                                      //default definition of strings to filter
+var availableFilterTerms = newArray("no filtering", "include", "exclude");   //dont change this
+var filterTerms = newArray(filterStrings.length); for  (i = 0; i < filterStrings.length; i++) {filterTerms[i] = "no filtering";} //default definition of filter types (automatic)
+//var filterTerms = newArray("no filtering", "no filtering", "no filtering");  //default definition of filter types (manual)
+var displayFileList = false;                                                 //shall array window be shown? 
+*/
+function setDialogImageFileFilter() {
+Dialog.create("Image file filter...");  //enable use interactivity
+Dialog.addMessage("Define the file types to be processed:");
+Dialog.addString("Files should have this extension:", fileExtension);	//add extension
+//Dialog.addMessage("Define filter for files:");
+//for (i = 0; i < filterStrings.length; i++) {
+//	Dialog.addString((i + 1) + ") Filter this text from file list: ", filterStrings[i]);	
+//	Dialog.addChoice((i + 1) + ") Files with text are included/excluded?", availableFilterTerms, filterTerms[i]);	
+//	}
+//Dialog.addCheckbox("Show the file list to check?", displayFileList);	//if check file lists will be displayed
+Dialog.addMessage("This macro will make a new folder with a TIFF suffix where converted files are saved");
+Dialog.show();
+fileExtension = Dialog.getString();
+//for (i = 0; i < filterStrings.length; i++) {
+//	filterStrings[i] = Dialog.getString();	
+//	filterTerms[i] = Dialog.getChoice();	
+//	}
+//displayFileList = Dialog.getCheckbox();
+}
+
+
+var fileExtension = ".nd2";
+//var filterStrings = newArray("MAX","Processed");  
+//var availableFilterTerms = newArray("no filtering", "include", "exclude");   //dont change this
+//var filterTerms = newArray(filterStrings.length); for  (i = 0; i < filterStrings.length; i++) {filterTerms[i] = "no filtering";} 
+//var displayFileList = false;    
+  
+
+
+
+
+setDialogImageFileFilter();
+
+
+run("Bio-Formats Macro Extensions");
+
+
+dir1 = getDirectory("Choose folder with image files ");
+list = getFileList(dir1);
+
+setBatchMode(true);
+
+// create folders for the tifs
+	dir1parent = File.getParent(dir1);
+	dir1name = File.getName(dir1);
+	dir2 = dir1parent+File.separator+dir1name+"_Tiffs";
+	if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for tiff
+		}
+ 
+for (i=0; i<list.length; i++) {
+	showProgress(i+1, list.length);
+	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+	filename = dir1 + list[i];
+		if (endsWith(filename, fileExtension)){
+//if (endsWith(filename, "czi")){
+		path=dir1+list[i];
+
+	//how many series in this czi file?
+		Ext.setId(path);//-- Initializes the given path (filename).
+		Ext.getSeriesCount(seriesCount); //-- Gets the number of image series in the active dataset.
+	
+		for (j=1; j<=seriesCount; j++) {
+		
+		run("Bio-Formats", "open=path autoscale color_mode=Default view=Hyperstack stack_order=XYCZT series_"+j);
+	
+		name=File.nameWithoutExtension;
+		seriesname=name;
+	//	saveAs("/Users/glyn_nelson/Desktop/test/cziTest/heart_point3airy_decon_with_measured_PSF-2.tif");
+	//	saveAs("TIFF", dir2+File.separator+name+"_"+j+".tif");
+		run("OME-TIFF...", "save=" + dir2+File.separator+name+"_"+j+".ome.tif" + " compression=Uncompressed");
+		run("Close All");
+		
+	}
+
+}
+run("Close All");
+setBatchMode(false);
+
+}
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
+ // macro
+
+
+
+
+
diff --git a/convert_to_tiff_with choose_filetype.ijm b/convert_to_tiff_with choose_filetype.ijm
new file mode 100644
index 0000000..3292847
--- /dev/null
+++ b/convert_to_tiff_with choose_filetype.ijm	
@@ -0,0 +1,101 @@
+//FUNCTION: opens a dialog to set text list for filtering a list
+//example: setDialogImageFileFilter();
+//this function set interactively the global variables used by the function getFilteredFileList
+//it can be set to ask for other text to filter files on.  I didn't get this to work though and have removed them.
+//this function needs global variables! (see below)
+/*
+
+var fileExtension = ".tif";   												//default definition of extension
+var filterStrings = newArray("","","");                                      //default definition of strings to filter
+var availableFilterTerms = newArray("no filtering", "include", "exclude");   //dont change this
+var filterTerms = newArray(filterStrings.length); for  (i = 0; i < filterStrings.length; i++) {filterTerms[i] = "no filtering";} //default definition of filter types (automatic)
+//var filterTerms = newArray("no filtering", "no filtering", "no filtering");  //default definition of filter types (manual)
+var displayFileList = false;                                                 //shall array window be shown? 
+*/
+function setDialogImageFileFilter() {
+Dialog.create("Image file filter...");  //enable use interactivity
+Dialog.addMessage("Define the file types to be processed:");
+Dialog.addString("Files should have this extension:", fileExtension);	//add extension
+//Dialog.addMessage("Define filter for files:");
+//for (i = 0; i < filterStrings.length; i++) {
+//	Dialog.addString((i + 1) + ") Filter this text from file list: ", filterStrings[i]);	
+//	Dialog.addChoice((i + 1) + ") Files with text are included/excluded?", availableFilterTerms, filterTerms[i]);	
+//	}
+//Dialog.addCheckbox("Show the file list to check?", displayFileList);	//if check file lists will be displayed
+Dialog.addMessage("This macro will make a new folder with a TIFF suffix where converted files are saved");
+Dialog.show();
+fileExtension = Dialog.getString();
+//for (i = 0; i < filterStrings.length; i++) {
+//	filterStrings[i] = Dialog.getString();	
+//	filterTerms[i] = Dialog.getChoice();	
+//	}
+//displayFileList = Dialog.getCheckbox();
+}
+
+
+var fileExtension = ".nd2";
+//var filterStrings = newArray("MAX","Processed");  
+//var availableFilterTerms = newArray("no filtering", "include", "exclude");   //dont change this
+//var filterTerms = newArray(filterStrings.length); for  (i = 0; i < filterStrings.length; i++) {filterTerms[i] = "no filtering";} 
+//var displayFileList = false;    
+  
+
+
+
+
+setDialogImageFileFilter();
+
+
+run("Bio-Formats Macro Extensions");
+
+
+dir1 = getDirectory("Choose folder with image files ");
+list = getFileList(dir1);
+
+setBatchMode(true);
+
+// create folders for the tifs
+	dir1parent = File.getParent(dir1);
+	dir1name = File.getName(dir1);
+	dir2 = dir1parent+File.separator+dir1name+"_Tiffs";
+	if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for tiff
+		}
+ 
+for (i=0; i<list.length; i++) {
+	showProgress(i+1, list.length);
+	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+	filename = dir1 + list[i];
+		if (endsWith(filename, fileExtension)){
+//if (endsWith(filename, "czi")){
+		path=dir1+list[i];
+
+	//how many series in this czi file?
+		Ext.setId(path);//-- Initializes the given path (filename).
+		Ext.getSeriesCount(seriesCount); //-- Gets the number of image series in the active dataset.
+	
+		for (j=1; j<=seriesCount; j++) {
+		
+		run("Bio-Formats", "open=path autoscale color_mode=Default view=Hyperstack stack_order=XYCZT series_"+j);
+	
+		name=File.nameWithoutExtension;
+		seriesname=name;
+	//	saveAs("/Users/glyn_nelson/Desktop/test/cziTest/heart_point3airy_decon_with_measured_PSF-2.tif");
+		saveAs("TIFF", dir2+File.separator+name+"_"+j+".tif");
+	//	run("OME-TIFF...", "save=" + dir2+File.separator+name+"_"+j+".tif" + " compression=Uncompressed");
+		run("Close All");
+		
+	}
+
+}
+run("Close All");
+setBatchMode(false);
+
+}
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
+ // macro
+
+
+
+
+
diff --git a/extract_qptiff_IMAGES_whole_dir.ijm b/extract_qptiff_IMAGES_whole_dir.ijm
new file mode 100644
index 0000000..e9c321d
--- /dev/null
+++ b/extract_qptiff_IMAGES_whole_dir.ijm
@@ -0,0 +1,51 @@
+//Writtent o save qptiff images as a tiff fomrat that will open without biorformats, for example, to import into Zen
+//This extratcs the first image in the qptiff, which has a pyramidal structure for fast loading- the first image is the
+//full resolution one.
+//The macro asks for the input directory and creates an output directory in the same parent folder,
+//with the same name plus the suffix _Tiffs
+
+//This will do all qptiff files in that directory for you.
+
+//I cannot get it to remove the filename suffix- I think the 5 char length is confusing the command I have tried.
+
+dir1 = getDirectory("Choose Source Directory ");
+dir1parent = File.getParent(dir1);
+dir1name = File.getName(dir1);
+dir2 = dir1parent+File.separator+dir1name+"_Tiffs";
+
+if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for tiff
+		}
+		
+//dir2 = getDirectory("Choose Destination Directory ");
+list = getFileList(dir1);
+setBatchMode(true);
+for (i=0; i<list.length; i++) {
+	 showProgress(i+1, list.length);
+	 filename = dir1 + list[i];
+	 name=File.nameWithoutExtension;
+	 if (endsWith(filename, "qptiff")) {
+//		 open(filename);
+//run("Bio-Formats Importer", "open="+filename+" autoscale color_mode=Custom rois_import=[ROI manager] view=Hyperstack stack_order=XYCZT series_0_channel_0_red=0 series_0_channel_0_green=0 series_0_channel_0_blue=255 series_0_channel_1_red=0 series_0_channel_1_green=255 series_0_channel_1_blue=0 series_0_channel_2_red=255 series_0_channel_2_green=0 series_0_channel_2_blue=0 series_0_channel_3_red=255 series_0_channel_3_green=255 series_0_channel_3_blue=255 view=Hyperstack stack_order=XYCZT"); 
+run("Bio-Formats Importer", "open=["+filename+"] autoscale color_mode=Default rois_import=[ROI manager] view=Hyperstack stack_order=XYCZT series_1");
+ // INSERT MACRO HERE
+title = getTitle();
+
+//	rename("original");
+//	Stack.getDimensions(width, height, channels, slices, frames);
+//	zstack=slices; 
+//wait(2000);
+//	run("Z Project...", "start=1 stop=slices projection=[Max Intensity]");
+
+//	selectWindow("MAX_original");
+	Stack.setDisplayMode("composite");
+//		 saveAs("TIFF", dir2+File.separator+list[i]+".tif");
+		  saveAs("TIFF", dir2+File.separator+name+".tif");
+//		 close();		
+close();
+ }
+}
+
+
+print("well, I think we gave them a damn");
+print("good thrashing there, what what?");
diff --git a/flatten_CZI_IMAGES_whole_dir.txt b/flatten_CZI_IMAGES_whole_dir.txt
new file mode 100644
index 0000000..ea758fc
--- /dev/null
+++ b/flatten_CZI_IMAGES_whole_dir.txt
@@ -0,0 +1,38 @@
+// modified with help from http://www.sussex.ac.uk/gdsc/intranet/pdfs/ImageJBatchProcessing.pdf
+// now doesn't care how many channels there are.  Just need to find a way to read the number of zeds too.
+//asks for input and output directories.  Best to create a separate output dir, but not necessary with the zvi if statement.
+//  In fact, could just change it....  DONE, now only want one dir for input and output.
+//Found it!  I can import the open image dimensions and specify the endpoitn for the MIP based on the number of slices!
+
+
+dir1 = getDirectory("Choose Source Directory ");
+//dir2 = getDirectory("Choose Destination Directory ");
+list = getFileList(dir1);
+setBatchMode(true);
+for (i=0; i<list.length; i++) {
+	 showProgress(i+1, list.length);
+	 filename = dir1 + list[i];
+	 if (endsWith(filename, "zvi")) {
+//		 open(filename);
+run("Bio-Formats Importer", "open="+filename+" autoscale color_mode=Custom rois_import=[ROI manager] view=Hyperstack stack_order=XYCZT series_0_channel_0_red=0 series_0_channel_0_green=0 series_0_channel_0_blue=255 series_0_channel_1_red=0 series_0_channel_1_green=255 series_0_channel_1_blue=0 series_0_channel_2_red=255 series_0_channel_2_green=0 series_0_channel_2_blue=0 series_0_channel_3_red=255 series_0_channel_3_green=255 series_0_channel_3_blue=255") view=Hyperstack stack_order=XYCZT"); 
+
+ // INSERT MACRO HERE
+title = getTitle();
+
+	rename("original");
+	Stack.getDimensions(width, height, channels, slices, frames);
+	zstack=slices; 
+//wait(2000);
+	run("Z Project...", "start=1 stop=slices projection=[Max Intensity]");
+
+	selectWindow("MAX_original");
+	Stack.setDisplayMode("composite");
+		 saveAs("TIFF", dir1+list[i]);
+		 close();
+		 close();
+ }
+}
+
+
+print("well, I think we gave them a damn");
+print("good thrashing there, what what?");
diff --git a/flatten_ZVI_IMAGES_whole_dir.txt b/flatten_ZVI_IMAGES_whole_dir.txt
new file mode 100644
index 0000000..2e0ce70
--- /dev/null
+++ b/flatten_ZVI_IMAGES_whole_dir.txt
@@ -0,0 +1,38 @@
+// modified with help from http://www.sussex.ac.uk/gdsc/intranet/pdfs/ImageJBatchProcessing.pdf
+// now doesn't care how many channels there are.  Just need to find a way to read the number of zeds too.
+//asks for input and output directories.  Best to create a separate output dir, but not necessary with the zvi if statement.
+//  In fact, could just change it....  DONE, now only want one dir for input and output.
+//Found it!  I can import the open image dimensions and specify the endpoitn for the MIP based on the number of slices!
+
+
+dir1 = getDirectory("Choose Source Directory ");
+//dir2 = getDirectory("Choose Destination Directory ");
+list = getFileList(dir1);
+setBatchMode(true);
+for (i=0; i<list.length; i++) {
+	 showProgress(i+1, list.length);
+	 filename = dir1 + list[i];
+	 if (endsWith(filename, "czi")) {
+//		 open(filename);
+run("Bio-Formats Importer", "open="+filename+" autoscale color_mode=Custom rois_import=[ROI manager] view=Hyperstack stack_order=XYCZT series_0_channel_0_red=0 series_0_channel_0_green=0 series_0_channel_0_blue=255 series_0_channel_1_red=0 series_0_channel_1_green=255 series_0_channel_1_blue=0 series_0_channel_2_red=255 series_0_channel_2_green=0 series_0_channel_2_blue=0 series_0_channel_3_red=255 series_0_channel_3_green=255 series_0_channel_3_blue=255 view=Hyperstack stack_order=XYCZT"); 
+
+ // INSERT MACRO HERE
+title = getTitle();
+
+	rename("original");
+	Stack.getDimensions(width, height, channels, slices, frames);
+	zstack=slices; 
+//wait(2000);
+	run("Z Project...", "start=1 stop=slices projection=[Max Intensity]");
+
+	selectWindow("MAX_original");
+	Stack.setDisplayMode("composite");
+		 saveAs("TIFF", dir1+list[i]);
+		 close();
+		 close();
+ }
+}
+
+
+print("well, I think we gave them a damn");
+print("good thrashing there, what what?");
diff --git a/foci_and_coloc_counting_wholedir_20x_moments_thresh.txt b/foci_and_coloc_counting_wholedir_20x_moments_thresh.txt
new file mode 100644
index 0000000..603ad9a
--- /dev/null
+++ b/foci_and_coloc_counting_wholedir_20x_moments_thresh.txt
@@ -0,0 +1,107 @@
+//Assumes the image is a MIP'ed tif with PML in channel1, H2AX in channel2 
+//(although these can be the other way round and it doesn't matter too much except for labelling in results)
+//and DAPI in channel3.  It looks for pure overlap and counts respective spots.
+//All results get stored in a concatenating results file.
+//Plus the mask images are stored (all 3 original images converted to masks plus the coloc maskin the 4th channel)
+// so you can see what it did.
+
+var tolerance = 30;
+run("Set Measurements...", "area integrated limit display redirect=None decimal=3");
+
+dir1 = getDirectory("Choose folder with tif files ");
+list = getFileList(dir1);
+
+setBatchMode(true);
+
+// create folders for the tifs
+	dir1parent = File.getParent(dir1);
+	dir1name = File.getName(dir1);
+	dir2 = dir1parent+File.separator+dir1name+"_masks";
+	if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for mask images
+		}
+
+for (i=0; i<list.length; i++) {
+	showProgress(i+1, list.length);
+	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+	filename = dir1 + list[i];
+	if (endsWith(filename, "tif")) {
+		path=dir1+list[i];
+		run("Bio-Formats Importer", "open="+filename+" autoscale color_mode=Default view=Hyperstack stack_order=XYCZT"); 
+		originalImage = getTitle();
+		roiManager("Reset");
+		selectWindow(originalImage);
+		run("Duplicate...", "duplicate channels=1"); //Change this to the DAPI channel slice*******
+//		run("8-bit");
+		setAutoThreshold("Moments dark");
+		run("Threshold");
+		run("Watershed");
+//		run("Entropy Threshold");
+		run("Analyze Particles...", "size=120-2500 circularity=0.04-1.00 exclude add");
+		rename("nuc");
+//nuclei now added to ROI manager
+
+//next find pml bodies
+		selectWindow(originalImage);
+		run("Duplicate...", "title=PML duplicate channels=3");
+//		run("8-bit");
+		setAutoThreshold("Triangle dark");
+		run("Threshold");
+//		rename("PML");
+
+//next find h2ax foci
+		selectWindow(originalImage);
+		run("Duplicate...", "title=h2ax duplicate channels=2");
+		wait(1000);
+//		run("8-bit");
+		setAutoThreshold("MaxEntropy dark");
+		run("Threshold");
+//		rename("h2ax");
+
+//Next find colocalised PML-H2AX foci
+		imageCalculator("Multiply create", "h2ax","PML");
+		selectWindow("Result of h2ax");
+		rename(originalImage+":coloc");
+		run("Grays");
+		for(j=0; j<roiManager("count"); j++) {
+			roiManager("select", j);
+			run("Find Maxima...", "noise="+tolerance+" output=[Count]");
+//	run("Find Maxima...", "noise="+tolerance+" output=[Point Selection]");
+			run("Add Selection...");
+			}
+//		saveAs("Results", dir2+File.separator+originalImage+"_nuc_coloc.xls"); 
+
+//next find H2AX foci per nuc
+		selectWindow("h2ax");
+		rename(originalImage+":h2ax");
+		for(j=0; j<roiManager("count"); j++) {
+			roiManager("select", j);
+			run("Find Maxima...", "noise="+tolerance+" output=[Count]");
+//	run("Find Maxima...", "noise="+tolerance+" output=[Point Selection]");
+			run("Add Selection...");
+			}
+		saveAs("Results", dir2+File.separator+originalImage+"_nuc_coloc_and_h2ax.xls"); //This is savign as it goes along in case it crashes
+		selectWindow(originalImage+"h2ax");
+		rename("h2ax");
+		selectWindow(originalImage+"coloc");
+		rename("coloc");
+		selectWindow(originalImage);
+		close();
+
+		run("Merge Channels...", "c1=PML c2=h2ax c3=nuc c4=coloc create");
+		rename("masks");
+
+		saveAs("TIFF", dir2+File.separator+originalImage+"_colocmasks.tif");
+
+
+	close();
+	}
+setBatchMode(false);
+}
+
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");
+
+// macro 
+
+
+
diff --git a/manual_randomSample_counting_WholeDir_manualSampling.ijm b/manual_randomSample_counting_WholeDir_manualSampling.ijm
new file mode 100644
index 0000000..8495ea7
--- /dev/null
+++ b/manual_randomSample_counting_WholeDir_manualSampling.ijm
@@ -0,0 +1,130 @@
+/* Updated March 2020.
+ * 
+ * Macro to go through a directory of images and open each one of the chosen filetype.
+ * It then asks afor a sampling frequency in x and y pixels- defaults to 200x200.
+ * These are then scaled to a 4k monitor resolution (think it fails if your image is larger than this)
+ * and a grid overlay added.
+ * Positive cells at each crosshair can then be counted using the mulitpoint tool
+ * 
+ * Outputs:
+ * An empty textfile with a filename sayign what the imagescaling is and the sampling frequency (requiring user to perform some
+ * basic maths).
+ * A csv file with each filename and the number of positive cells per sampling site per image
+ * 
+ * Note:
+ * occasionally it decides there are changes to the image to save- seems to want to save it with the ROI overlay once you have added more than 10 dots???.
+ * 
+ * Could have the samplingfreq and scaling in the results filename if I could guarantee that all images were the same scale.
+ */
+
+w = 4096;
+h = 2160;
+var xpoints = (0);
+var ypoints = (0);
+var count =(0);
+
+var fileExtension = ".tif"; //sets the default file type
+var DistanceSet = (200); //Sets the deault sampling frequency
+
+setDialogImageFileFilter();
+
+
+run("Bio-Formats Macro Extensions");
+
+
+//get user to decide sampling frwquency
+setSamplingFreq();
+SamplingArea= DistanceSet * DistanceSet;
+     dir = getDirectory("Choose Source Directory ");
+     list = getFileList(dir);
+     for (i=0; i<list.length; i++) {
+        showProgress(i+1, list.length);
+		print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+		filename = dir + list[i];
+		if (endsWith(filename, fileExtension)) {
+        	path=dir+list[i];
+//5 commented out lines below are for using raw files with multiple images in them, eg czi, lif etc.
+	//how many series in this file?
+//			Ext.setId(path);//-- Initializes the given path (filename).
+//			Ext.getSeriesCount(seriesCount); //-- Gets the number of image series in the active dataset.
+	
+//			for (j=1; j<=seriesCount; j++) {
+//				run("Bio-Formats", "open=path autoscale color_mode=Default view=Hyperstack stack_order=XYCZT series_"+j);		
+				run("Bio-Formats", "open=path autoscale color_mode=Default view=Hyperstack stack_order=XYCZT series_");
+	
+//       		 open(dir+list[i]);
+				name=getTitle();
+				getPixelSize(unit, pixelWidth, pixelHeight);
+				saveAs("Results", dir+File.separator+name+"_ScalingIs_"+pixelWidth+"_"+unit+"_samplingEvery"+DistanceSet+"pixels.txt");
+//				print("image scale is "+pixelWidth+" "+unit+" per pixel.  Sampling is every 200x200 pixels");
+				run("Canvas Size...", "width=&w height=&h position=Center zero");
+				run("Maximize");
+				setLocation(0,0);
+				run("Unsharp Mask...", "radius=1 mask=0.60");
+				run("Grid...", "grid=Crosses area=SamplingArea color=green bold center");
+				run("Set... ", "zoom=50");
+    		    setTool("multipoint");
+//2 lines below added to allow zeroes to be counted by tricking the counter using the addition of a single count in the xpoints which is then
+//subtracted at the point of reporting.
+				run("Point Tool...", "type=Dot color=Black size=[Tiny] counter=0");
+    		    makePoint(0, 0, "tiny black dot");
+				run("Point Tool...", "type=Dot color=Red size=[Extra Large] show counter=0");
+ //  		     name=File.nameWithoutExtension;				
+				waitForUser("Your Action Required", "Click on all crosshairs that are inside your positive signal area.\n Alt-Click on a spot to remove it.\n Then press OK");
+				getSelectionCoordinates( xpoints, ypoints );
+				count = (xpoints.length -1);
+//The if statement below doesn't work- clicking OK without markign nay points gives an error because the xpoints, ypoints array is empty.
+//This also means that the whole macro fails if there are no positives in a field.
+
+//		   if (selectionType() == -1) { // In case there might be zero points selected by the user
+//		     count = 0;
+//		   } else {
+//				getSelectionCoordinates(xpoints, ypoints);
+//				count = (xpoints.length -1);
+//				   }
+			row = nResults;
+			setResult("Label", row, getTitle());
+			setResult("Count", row, count);
+			close();
+			count = 0;
+     	}
+     }
+saveAs("Results", dir+File.separator+name+"_AllCountResults.csv");
+run("Clear Results");
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
+
+
+//FUNCTION- choose file type
+function setDialogImageFileFilter() {
+Dialog.create("Image file filter...");  //enable use interactivity
+Dialog.addMessage("Define the file types to be processed:");
+Dialog.addString("Files should have this extension:", fileExtension);	//add extension
+//Dialog.addMessage("Define filter for files:");
+//for (i = 0; i < filterStrings.length; i++) {
+//	Dialog.addString((i + 1) + ") Filter this text from file list: ", filterStrings[i]);	
+//	Dialog.addChoice((i + 1) + ") Files with text are included/excluded?", availableFilterTerms, filterTerms[i]);	
+//	}
+//Dialog.addCheckbox("Show the file list to check?", displayFileList);	//if check file lists will be displayed
+Dialog.addMessage("This macro will sequentially open all these filetypes in the chosen folder");
+Dialog.show();
+fileExtension = Dialog.getString();
+//for (i = 0; i < filterStrings.length; i++) {
+//	filterStrings[i] = Dialog.getString();	
+//	filterTerms[i] = Dialog.getChoice();	
+//	}
+//displayFileList = Dialog.getCheckbox();
+}
+//End of function setDialogImageFileFilter
+
+
+//FUNCTION- determine sampling frequency
+function setSamplingFreq() {
+Dialog.create("Set the sampling frequency");  //enable use interactivity
+Dialog.addMessage("Define the distance between points (in pixels):");
+Dialog.addString("Sampling Frequency should be this far apart:", DistanceSet);	//add extension
+Dialog.addMessage("This sets the horizontal and vertical distance between sampling points");
+Dialog.show();
+StringDistanceSet = Dialog.getString(); //Not sure this is needed in here as it seems to know numbers aren't strings in string boxes.
+DistanceSet = parseFloat(StringDistanceSet);
+}
+//End of function setSamplingFreq
\ No newline at end of file
diff --git a/manual_spot_coutning_WholeDir_example.ijm b/manual_spot_coutning_WholeDir_example.ijm
new file mode 100644
index 0000000..66a0c48
--- /dev/null
+++ b/manual_spot_coutning_WholeDir_example.ijm
@@ -0,0 +1,90 @@
+w = 4096;
+h = 2160;
+var xpoints = (0);
+var ypoints = (0);
+var count =(0);
+
+var fileExtension = ".jpg"; //sets the default file type
+
+setDialogImageFileFilter();
+
+
+run("Bio-Formats Macro Extensions");
+
+setBatchMode(true);
+
+     dir = getDirectory("Choose Source Directory ");
+     list = getFileList(dir);
+     for (i=0; i<list.length; i++) {
+        //showProgress(i+1, list.length);
+		//print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+		filename = dir + list[i];
+		if (endsWith(filename, fileExtension)){
+ //       	showProgress(i+1, list.length);
+        	path=dir+list[i];
+
+	//how many series in this file?
+			Ext.setId(path);//-- Initializes the given path (filename).
+			Ext.getSeriesCount(seriesCount); //-- Gets the number of image series in the active dataset.
+	
+			for (j=1; j<=seriesCount; j++) {
+		
+//				run("Bio-Formats", "open=path autoscale color_mode=Default view=Hyperstack stack_order=XYCZT series_"+j);
+	
+       		 open(dir+list[i]);
+	   		   run("Canvas Size...", "width=&w height=&h position=Center zero");
+      		  run("Maximize");
+   		     setLocation(0,0);
+ //    		   type = selectionType(10); 
+      		  run("Unsharp Mask...", "radius=1 mask=0.60");
+                  run("Grid...", "grid=Crosses area=85700 color=green bold center");
+     		   run("Set... ", "zoom=50");
+    		    setTool("multipoint");
+   		     run("Point Tool...", "type=Dot color=Red size=[Extra Large] label show counter=0");
+   		     name=File.nameWithoutExtension;
+ //       getSelectionCoordinates( xpoints, ypoints );
+ //           x = xpoints[0];
+ //   y = ypoints[0];
+//        count = xpoints.length;
+        row = nResults;
+//        setResult("Label", row, getTitle());
+		setResult("Label", row, filename);
+        setResult("Count", row, count);
+        waitForUser("Your Action Required", "Double click when center point of cross is inside,\n one click when on border or when have doubt.\n Then press OK");
+		          getSelectionCoordinates( xpoints, ypoints );
+		   if (selectionType() == -1) { // In case there might be zero points selected by the user
+		     count = 0;
+		   } else {
+		     getSelectionCoordinates(xpoints, ypoints);
+		     count = xpoints.length;
+				   }
+   row = nResults;
+   setResult("Label", row, getTitle());
+   setResult("Count", row, count);
+       close();
+     	}
+     }
+saveAs("Results", dir+File.separator+name+"_"+j+"_countResults".csv");
+
+
+//FUNCTION- choose file type
+function setDialogImageFileFilter() {
+Dialog.create("Image file filter...");  //enable use interactivity
+Dialog.addMessage("Define the file types to be processed:");
+Dialog.addString("Files should have this extension:", fileExtension);	//add extension
+//Dialog.addMessage("Define filter for files:");
+//for (i = 0; i < filterStrings.length; i++) {
+//	Dialog.addString((i + 1) + ") Filter this text from file list: ", filterStrings[i]);	
+//	Dialog.addChoice((i + 1) + ") Files with text are included/excluded?", availableFilterTerms, filterTerms[i]);	
+//	}
+//Dialog.addCheckbox("Show the file list to check?", displayFileList);	//if check file lists will be displayed
+Dialog.addMessage("This macro will make a new folder with a TIFF suffix where converted files are saved");
+Dialog.show();
+fileExtension = Dialog.getString();
+//for (i = 0; i < filterStrings.length; i++) {
+//	filterStrings[i] = Dialog.getString();	
+//	filterTerms[i] = Dialog.getChoice();	
+//	}
+//displayFileList = Dialog.getCheckbox();
+}
+//End of function filetype
diff --git a/measure_multiImage_CZI_5chMP.ijm b/measure_multiImage_CZI_5chMP.ijm
new file mode 100644
index 0000000..f81de08
--- /dev/null
+++ b/measure_multiImage_CZI_5chMP.ijm
@@ -0,0 +1,135 @@
+macro 'convert CZI to tiff' {
+
+/*
+ * - converts czi files into TIFF and analyses them. 
+ * - the macro ask for an input folder that should contain 1 or more czi files.
+ * - if they are tilescans, it saves each tile out as a tif and analyses independently.
+ * - each series is saved as one tiff. The name of the series is appended to the file name.
+ * - The TIFFs are saved in a folder generated by the macro in the same 
+ * - parent folder as the lifs, with the same prefix for the folder name, with TIFF suffix.
+ * - The analyses are stored in separate folders in a similar manner with the names _Areas
+ * - and _Objects
+ * 
+ */
+
+run("Bio-Formats Macro Extensions");
+
+
+dir1 = getDirectory("Choose folder with czi files ");
+list = getFileList(dir1);
+
+setBatchMode(true);
+
+// create folders for the tifs
+	dir1parent = File.getParent(dir1);
+	dir1name = File.getName(dir1);
+	dir2 = dir1parent+File.separator+dir1name+"_Tiffs";
+	dir3 = dir2+File.separator+"_Areas";
+	dir4 = dir2+File.separator+"_Objects";
+	if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for tiff
+		}
+	if (File.exists(dir3)==false) {
+				File.makeDirectory(dir3); // new directory for area analsysis
+		}
+	if (File.exists(dir4)==false) {
+				File.makeDirectory(dir4); // new directory for object analysis
+		}
+ 
+for (i=0; i<list.length; i++) {
+	showProgress(i+1, list.length);
+	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+	filename = dir1 + list[i];
+		if (endsWith(filename, "czi")){
+		path=dir1+list[i];
+
+	//how many series in this czi file?
+		Ext.setId(path);//-- Initializes the given path (filename).
+		Ext.getSeriesCount(seriesCount); //-- Gets the number of image series in the active dataset.
+	
+		for (j=1; j<=seriesCount; j++) {
+		
+		run("Bio-Formats", "open=path autoscale color_mode=Default view=Hyperstack stack_order=XYCZT series_"+j);
+	
+		name=File.nameWithoutExtension;
+		seriesname=name;
+	//	saveAs("/Users/glyn_nelson/Desktop/test/cziTest/heart_point3airy_decon_with_measured_PSF-2.tif");
+		saveAs("TIFF", dir2+File.separator+name+"#"+j+".tif");
+
+//Now analyse the image
+//modified for 4 channel images ch1 920 SHG, ch2 920 GFP, ch3 1040 SHG, ch4 1040 AF/vasc dye
+title =getTitle();
+rename("tilescan1cleaner_4wk");
+run("Make Composite");
+
+//first merge both shg as a minimum to remove shot noise
+run("Duplicate...", "title=shg1 duplicate channels=1");
+selectWindow("tilescan1cleaner_4wk") ;
+run("Duplicate...", "title=shg2 duplicate channels=4");
+
+imageCalculator("Min create", "shg1","shg2");
+selectWindow("Result of shg1");
+rename("SHG_" + title);
+run("Despeckle");
+run("Set Measurements...", "area mean standard perimeter integrated area_fraction limit display redirect=None decimal=3");
+run("Measure");
+close();
+selectWindow("shg1");
+close();
+selectWindow("shg2");
+close();
+
+//now clean up GFP using the AF in red channel
+selectWindow("tilescan1cleaner_4wk") ;
+run("Duplicate...", "title=green duplicate channels=2");
+//selectWindow("tilescan1cleaner_4wk") ;
+//run("Duplicate...", "title=greenBgrnd duplicate channels=3");
+//imageCalculator("Subtract create", "green","greenBgrnd");
+//selectWindow("Result of green");
+
+//setAutoThreshold("Huang dark");
+//run("Convert to Mask");
+//run("Close");
+rename("GFP_" + title);
+//setMinAndMax(1000, 4095);
+//run("Despeckle");
+//run("Apply LUT");
+run("Measure");
+//rename("binary");
+saveAs("Results", dir3+File.separator+name+"#"+j+"_GFPandColl_Area.csv");
+run("Clear Results");
+
+selectWindow("GFP_"+title);
+
+//setAutoThreshold("IsoData dark");
+
+//setAutoThreshold("Huang dark");
+//run("Convert to Mask"); //needs to be made binary for the dilate to run
+//run("Dilate");
+run("Gaussian Blur...", "sigma=4"); //obviously loses the binary-ness
+//setAutoThreshold("MaxEntropy dark");
+run("Convert to Mask");
+//run("Make Binary");
+//selectWindow("binary");
+rename("GFPbinary_" + title);
+run("Analyze Particles...", "size=30.00-Infinity display");
+//run("Analyze Particles...", "size=30-Infinity summarize");
+//SelectWindow(Summary);
+saveAs("Results", dir4+File.separator+name+"#"+j+"_GFP_Objects.csv");
+run("Clear Results");
+
+
+
+		run("Close All");
+//		run("Collect Garbage");
+		
+	}
+
+}
+//showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
+run("Close All");
+setBatchMode(false);
+
+}
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
+ // macro
diff --git a/per_nuc_FISH_modified_Tengfei.txt b/per_nuc_FISH_modified_Tengfei.txt
new file mode 100644
index 0000000..38b16b3
--- /dev/null
+++ b/per_nuc_FISH_modified_Tengfei.txt
@@ -0,0 +1,95 @@
+//Macro for FISH telomere intensity analysis by Glyn Nelson
+//Assumes you input an RGB image with the telomeres in the red channel and the nuclear counterstain in the green channel.
+//  This macro will identify bright objects in the red channel of an RGB image that are within the bright objects of the green channel
+//It assumes levels for the two thresholds used to determine the object sizes
+//would be better with a method of manual adjustment for the nuclear threshold
+
+// inputs for the macro
+ 
+    Dialog.create("Telomere Intensity measurement");
+    Dialog.addMessage("This macro measures telomere intensities per nucleus. ");
+ Dialog.addMessage("");   
+ Dialog.addMessage("It asks you to open an image- please open your image as a merged RGB image with the ");
+    Dialog.addMessage("telomeres in the red channel and the nuclear counterstain in the green channel. ");
+ Dialog.addMessage("");   
+    Dialog.addMessage("Data for telomere fluorescence per nucleus are then saved in an excel file in the same folder as the image file. ");
+
+    Dialog.show();
+
+open();
+title = getTitle();
+run("Split Channels");
+wait(140);
+close();
+wait(140);
+rename("nuc");
+run("Put Behind [tab]");
+wait(140);
+rename("foci");
+wait(140);
+run("Merge Channels...", "red=[foci] green=[nuc] blue=*None* gray=*None* keep");
+
+selectWindow("foci");
+run("Hi Lo Indicator");
+run("Brightness/Contrast...");
+Stack.setChannel(1);
+waitForUser("Please adjust ch1 if necessary, apply, then press OK here.");
+Stack.setChannel(2);
+waitForUser("Please adjust ch1 if necessary, apply, then press OK here.");
+//Dialog.create("User input required");
+//Dialog.addMessage("Please adjust minimum signal to remove non telomeric fluorescence if necessary, then press 'Apply'...");   
+//    Dialog.show();
+//print("Please adjust minimum signal to remove non telomereic fluorescence if necessary, then press 'Apply'...");
+//wait(12000);
+//run("Apply LUT");
+run("Put Behind [tab]");
+wait(140);
+
+selectWindow("RGB");
+
+selectWindow("nuc");
+//setAutoThreshold();
+run("Threshold...");
+waitForUser("Now please adjust threshold to highlight nuclear signal, apply the threshold, then press OK here,");
+//Dialog.create("User input required");
+//Dialog.addMessage("Now please adjust threshold to highlight nuclear signal, then press 'Apply'...");   
+//    Dialog.show();
+//print("Now please adjust threshold to highlight nuclear signal, then press 'Apply'...");
+//wait(12000);
+//setThreshold(102, 255);
+run("Fill Holes");
+
+setForegroundColor(0, 0, 0);
+run("Invert LUT");
+setTool(20);
+//this is pointing to a specific button in the gui for flood fill.  Had to add in lut inverter to get tit to work- don't understand that.
+
+//Dialog.create("User input required");
+//Dialog.addMessage("Please remove unwanted nuclei by clicking on them");
+//Dialog.addMessage("- ignore small spots and those on the edge...");   
+//    Dialog.show();
+waitForUser("Please remove unwanted nuclei by clicking on them- ignore small spots and those on the edge.  Click OK when finished");
+//wait(30000);
+run("Convert to Mask");
+wait(140);
+
+run("Analyze Particles...", "size=20-300 circularity=0.00-1.00 show=Nothing exclude add");
+
+run("Put Behind [tab]");
+selectWindow("foci");
+wait(140);
+rename(title);
+run("Set Measurements...", "display area integrated redirect=None decimal=3");
+roiManager("Measure");
+String.copyResults();
+roiManager("Delete");
+selectWindow("Results");
+saveAs("Measurements", getDirectory("current")+"FISHanalysis_per_nuc"+ substring(title,0) +".xls");
+
+close();
+close();
+close();
+
+run("Clear Results");
+
+
diff --git a/realign_20x_DM5500_Cy5cube.ijm b/realign_20x_DM5500_Cy5cube.ijm
new file mode 100644
index 0000000..68c541b
--- /dev/null
+++ b/realign_20x_DM5500_Cy5cube.ijm
@@ -0,0 +1,82 @@
+//To relaign the Y5 cube to the new Cy3 cube on the DM5500 20x, by moving the 4th channel up 4 pixels. 
+run("Bio-Formats Macro Extensions");
+
+
+dir1 = getDirectory("Choose folder with lif files ");
+list = getFileList(dir1);
+
+setBatchMode(true);
+
+// create folders for the tifs
+	dir1parent = File.getParent(dir1);
+	dir1name = File.getName(dir1);
+	dir2 = dir1parent+File.separator+dir1name+"_Tiffs";
+	if (File.exists(dir2)==false) {
+				File.makeDirectory(dir2); // new directory for tiff
+		}
+ 
+for (i=0; i<list.length; i++) {
+	showProgress(i+1, list.length);
+	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
+	filename = dir1 + list[i];
+		if (endsWith(filename, "lif")){
+		path=dir1+list[i];
+
+	//how many series in this lif file?
+		Ext.setId(path);//-- Initializes the given path (filename).
+		Ext.getSeriesCount(seriesCount); //-- Gets the number of image series in the active dataset.
+	
+		for (j=1; j<=seriesCount; j++) {
+		
+		run("Bio-Formats", "open=path autoscale color_mode=Default view=Hyperstack stack_order=XYCZT series_"+j);
+	
+		name=File.nameWithoutExtension;
+
+	//retrieve name of the series from metadata
+		text=getMetadata("Info");
+		n1=indexOf(text," Name = ")+8;// the Line in the Metadata reads "Series 0 Name = ". Complete line cannot be taken, because
+									  // The number changes of course. But at least in the current version of Metadata this line is the 
+									  // only occurence of " Name ="
+		n2=indexOf(text,"SizeC = ");  // this is the next line in the Metadata
+		extractedname=substring(text,n1,n2-2);
+		cleanname=split(extractedname,"/");  // this is done here to remove the forward slash that is found in the image name 
+					// if it is part of a mark and find subfolder, for example 
+
+//We still want both parts of the filenename.  So if there are two parts to the array, then stick them together, otherwise just take the first:
+			if (cleanname.length==2){
+			seriesname=cleanname[0]+cleanname[1];
+			}		
+			else seriesname=cleanname[0];
+
+
+			originalImage = getTitle();
+			selectWindow(originalImage);
+//Make a copy of each channel since the Translate option will move all channels in a multichannel image
+			run("Duplicate...", "title=green duplicate channels=1"); 
+			selectWindow(originalImage);
+			run("Duplicate...", "title=red duplicate channels=2"); 
+			selectWindow(originalImage);
+			run("Duplicate...", "title=blue duplicate channels=3"); 
+			selectWindow(originalImage);
+			run("Duplicate...", "title=farred duplicate channels=4"); 
+//Assuming that channel 1 is green, 2 is red, 3 is blue and 4 is far red
+			selectWindow("farred");
+//Translate Cy5 image relative to the red (Cy3 cube) image:
+			run("Translate...", "x=0 y=3 interpolation=None stack");
+
+//Re-merge the channels:
+			run("Merge Channels...", "c1=green c2=red c3=blue c4=farred create");
+
+//Save image out as tiff:
+			saveAs("TIFF", dir2+File.separator+name+"_"+seriesname+".tif");
+			run("Close All");
+		
+	}
+
+}
+showMessage(" Well, I think we gave them a damn good thrashing there, what what??");	
+run("Close All");
+setBatchMode(false);
+
+
+