Calculation for FRET Maps.

FRET MAP

macro "Ratiometric FRET" {

path    = getDirectory("Choose a Directory");
list    = getFileList(path);
length  = list.length;

File.makeDirectory(path + "Imaging/");

//FRET map creation

for (i=0; i<length; i++) {
             
               n=i+1;
               open(path+list[i]);
               img = File.nameWithoutExtension();
               selectWindow(img+".lsm");
               run("Subtract Background...", "rolling=300 stack");
               run("StackReg", "transformation=Translation");
               run("Median...", "radius=1.3 stack");
               run("Stack to Images");
               run("Duplicate...", "title=mask");
               run("Threshold...");
               setAutoThreshold("Default dark");
               waitForUser("Imagen "+n+" de "+length,"Apply Threshold y click OK");
               run("16-bit");
               run("Divide...", "value=255");
               saveAs("Tiff", path + "Imaging/mask 000" + i);
               imageCalculator("Divide create 32-bit", "1-0002","1-0001");
               imageCalculator("Multiply create","Result of 1-0002","mask 000"+i+".tif");
               selectWindow("Result of Result of 1-0002");
               saveAs("Tiff", path + "Imaging/fret map " + i);

run("Close All");

}

Hello,
This is the description that what I want to do:
1 Open the files from folder
2 Subtract background to all the channels (300 pixels)
3 Apply median filter to all channels (1.3 pixels)
4 Align channels with pluging “registration”- Stackreg- using the “Translation mode”
5 Split the channels or stack to image
6 Duplicate C1 (channel 1) or image 1 (CFP)
7 Create a binary mask of CFP image with the Threshold tool. (MASK channel 1)
8 Use the tool math, divide the binary mask by 255.
9 Use the tool image calculator: divide C2 (channel 2 or C2 = FRET) by C1 (channel 1 or C1 = CFP) Press 32 bit in the options.
10 Finally, multiply the MASK channel 1 by the result of step 9
11 save image in a new folder in Tiff

I want to make this macro generic for any image name ( I have my images in a folder with numbers from 1 to 10 or more. However, I do not know how to make this generic so then any number or name can be detected and continue with the macro.
I receive this kind of error message in the macro.

Hope can you help me with this doubt,

Thanks a lot,

Kind Regards

Pablo

line 20

   run("Stack to Images");

If you want to split channels (step 5), it's much better to use the command [Image > Color Split Chanells], which is

   run("Split Channels");

In this way, images after splitting will have suffix "C1-" for the first channel, "C-2" for the second channel... and so on. For this reason, the name of these windows for each single channel becomes

"C1-" + list[ i ]

For the first channel,

"C2" +list[i]

Or. this might be what you want to do?

---

By the way,

line 14-16

    open(path+list[i]);
    img = File.nameWithoutExtension();
    selectWindow(img+".lsm");

If the original file name is with .lsm, I just wonder if there is any specific reason for this.

Just to be tidy and better organization, indent lines within a macro. See here

Thank you very much, Kota
the .lsm is because my images are from Zeiss microscope, but I would like to do that generic too

Follow your suggestion, I will use run("Split Channels");

So I wrote again the lines 28-30 , now after Split Channels.

          28  imageCalculator("Divide create 32-bit", "C2-" + list[ i ], "C1-" + list[ i ]");
          29   imageCalculator("Multiply create","C2-" + list[ i ]","mask 000"+i+".tif");
          30  selectWindow("Result of Result of "C2-" + list[ i ]");

However, I am getting this error

How can I fix it? I try to change the " " but nothing works.

Thanks for any help.

So the problem is this line

selectWindow("Result of Result of "C2-" + list[ i ]");

Too many double quoting! It should be enough with

selectWindow("Result of Result of C2-" + list[ i ]);