/* * Macro to process a folder of LIFs into Channel Split and Channel Merged (Projected) images * * #### Change Log #### * v0.1 - 1/23/20 - Initial Version - CK * * v0.2 - 1/30/20 - Changed script to split into up to 4 channels instead of RGB. * Fixed handling of 2 channel images by changing the order in which channel colors are set. - CK * * v0.3 - 2/13/20 - Changed how the script handles saving files. * Added in the ability to pick what colors you want each channel to be. Note the default order is Blue,Green,Red,Cyan. * This script is also limited to handling only 4 channel images. If you somehow managed to image more channels than * that let me know and share with me a test file and we can fix it. * Added better dialog when running the script to make it as clear and easy to use as I can think of * Added a parameter to allow for either a flat single channel RGB output or a multichannel tif output-- CK * * v0.4 - 2/14/20 - Added in an option to use Average Intensity Projection as well as Max Intensity Projection * Added in an option to not project, useful to simply seperate out a lif into z-stacks tifs and set the colors. Still will be easier to work with the files and rename them. * Added all the options for projections to the script * Added a failsafe mode for when the user tries to split the Z-Stack into colors without projecting and trying to save as Flat images, now it will simply split the channels without projecting. * Changed the color suffixes added to the filenames to be 2 letters, for clarity. */ macro "LIFs to TIFs" { #@ String (visibility=MESSAGE, value="

How to use:

Select the folder with the LIF files you want to process.
Check the project box and select the type of projection you want to do.
- If it is unchecked you are simply seperating the LIF to make it easier to work with.
- If you are not projecting you can ignore the drop down box.
Check Split Colors if you want to split into individual channels.
Check TIFF Stack box if you want a multi-channel tiff as your maximum intensity projection output.
- Otherwise the output is a RGB image where adjusting the channel colors will be harder.
Pick the colors you want for the corresponding channels.
- Do not worry about channels you do not have, if they don't exist in the LIF they will not be in the output.
The output will be in a folder that is named the same as the input folder but with '--Tiff' at the end.

", required=false) msg #@ File (label = "Directory with LIF file(s):", style = "directory") input #@ Boolean (label="Check to project:", value=true, persist=false) should_proj #@ String (label = "Projection Type:", choices={"Max Intensity", "Average Intensity", "Min Intensity", "Sum Slices", "Standard Deviation", "Median"}, style="listBox", value="Max Intensity", persist=false) proj_type #@ Boolean (label = "Check to Split Colors:", persist=false, value=false ) split_colors #@ Boolean (label = "TIFF Stack:", persist=false, value = false) tiff_stack #@ String (label = "Channel 1 color:", choices={"Blue", "Red","Green","Cyan","Magenta","Yellow", "Grays"}, style="listBox", value="Blue", persist=false) channel1_color #@ String (label = "Channel 2 color:", choices={"Blue", "Red","Green","Cyan","Magenta","Yellow", "Grays"}, style="listBox", value="Green", persist=false) channel2_color #@ String (label = "Channel 3 color:", choices={"Blue", "Red","Green","Cyan","Magenta","Yellow", "Grays"}, style="listBox", value="Red", persist=false) channel3_color #@ String (label = "Channel 4 color:", choices={"Blue", "Red","Green","Cyan","Magenta","Yellow", "Grays"}, style="listBox", value="Cyan", persist=false) channel4_color // required for naming the color splits, as imageJ1macro does not support string indexing eq. string[0] channel1_color_first = substring(channel1_color, 0, 2); channel2_color_first = substring(channel2_color, 0, 2); channel3_color_first = substring(channel3_color, 0, 2); channel4_color_first = substring(channel4_color, 0, 2); if (tiff_stack == 0 && split_colors == 1 && should_proj == 0) tiff_stack = 1 ;{} //cant split colors without Projecting unless you are keeping TIF stacks run("Bio-Formats Macro Extensions"); suffix = ".lif"; //global variable thats used inside processFolder(input) setBatchMode(true); processFolder(input); run("Collect Garbage"); //It seems like imageJ isnt gracefully saving things without this. showMessage(" -- Finished! --"); // this allows the user to know when they can look at their files run("Close All"); Ext.close(); setBatchMode(false); // function to scan folders/subfolders/files to find files with correct suffix function processFolder(input) { list = getFileList(input); list = Array.sort(list); inputParent = File.getParent(input); inputName = File.getName(input); // create folders for the tifs output = inputParent+File.separator+inputName+"--Tiff"; if (File.exists(output)==false) { File.makeDirectory(output); // new directory for tiffs } for (k = 0; k < list.length; k++) { if(File.isDirectory(input + File.separator + list[k])) processFolder(input + File.separator + list[k]); if(endsWith(list[k], suffix)) lifToTiffRGBSplit(input, output, list[k]); showProgress(k+1, list.length); } } function splitColors() { if( split_colors == 1 ){ run("Duplicate...", "duplicate"); mergeChannelsFnc(); run("Split Channels"); for (i=1;i<=channels;i++) { selectImage(i); out_path = output + File.separator+name+"_"+seriesname+"_MIP_"+j +" " + color_name[i] +".tif"; saveAs("Tiff", out_path); } } else { mergeChannelsFnc(); } } function mergeChannelsFnc() { if (tiff_stack == 1) { //if (should_proj == 1) { // lets you keep the different channels if projected for the final merged flat image. //run("Make Composite"); saveAs("Tiff", output+File.separator+name+"_"+seriesname+"_MIP_"+j+".tif"); close(); //} else { // this gives us RGB Z stacks output for non-max projected, stacks. essentially just the output of the //run("RGB Color","slices"); //saveAs("Tiff", output+File.separator+name+"_"+seriesname+"_MIP_"+j+".tif"); //close(); //} } else { //this is where i need the logic to fix making tif RGB stacks tiff==1, split_colors==0, Proj == 0 // make a for loop to split each z step, run RGB TRYING run("RGB Color", slices) first run("RGB Color"); saveAs("Tiff", output+File.separator+name+"_"+seriesname+"_MIP_"+j+".tif"); close(); close(); } } function setChannelColors(){ //This function allows the user to set the colors. It is done in reverse order so that if there are less than 4 colors they are still assigned correctly Stack.setChannel(4); run(channel4_color); Stack.setChannel(3); run(channel3_color); Stack.setChannel(2); run(channel2_color); Stack.setChannel(1); run(channel1_color); } //"", , "Standard Deviation", function whichProjToDo() { if (should_proj == 1) { run("Z Project...", "projection=[" + proj_type + "]"); if( proj_type == "Max Intensity"){ proj_prefix = "MAX_"; } else if (proj_type == "Average Intensity") { proj_prefix = "AVG_"; } else if (proj_type == "Min Intensity"){ proj_prefix = "MIN_"; } else if (proj_type == "Sum Slices"){ proj_prefix = "SUM_"; } else if (proj_type == "Standard Deviation"){ proj_prefix = "STD_"; } else if (proj_type == "Median"){ proj_prefix = "MED_"; } return proj_prefix; } else { return 0; } } function lifToTiffRGBSplit(input, output, file) { color_name = newArray(" ", channel1_color_first, channel2_color_first, channel3_color_first , channel4_color_first); // need this for naming the output path= input + File.separator + list[k]; //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. //this is the loop that processes all the images WITHIN the LIF file. 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 seriesname=substring(text, n1, n2-2); seriesname=replace(seriesname,"/","-"); rename(seriesname); getDimensions(w, h, channels, slices, frames); //needed to we know how many channels we are working with, lets the logic work below. //project and save logic if(Stack.isHyperstack) { proj_prefix = whichProjToDo(); if (nSlices>1) Stack.setDisplayMode("composite");{ selectWindow(seriesname); if (should_proj == 1) { close(); //closes non-projected window if the projection was made selectWindow(proj_prefix + seriesname);//selects the projection and works with that. Not needed if not projecting anything } setChannelColors(); splitColors(); } } else if (nSlices>1) { if (nSlices>1) Stack.setDisplayMode("composite");{ setChannelColors(); splitColors(); } } else { Stack.setDisplayMode("composite"); setChannelColors(); splitColors(); } run("Close All"); run("Collect Garbage"); } Ext.close(); } }