I have hundreds of LSM files from a zeiss microscope. Each one is a 3D confocal image time point. The software should automatically concatenate these into a single file, but it didn't this time. It does it fairly quickly, but there appears to be no way to do this with data already saved into individual files.
I can open LSM files in imaris/fiji, but only one at a time. I can open a series, but imaris actually has to open all of them and crashes due to lack of RAM.
Even breaking it up into bits, there doesn't seem to be a way to put them into one file. I click stack, save images to stack, and it says something along the lines of "there must be at least two images open" despite the fact that there are dozens.
In FIJI you can go to Image -> Stacks -> Tools -> Concatenate and order your open files into a single, T-stacked file.
Here is a macro I wrote for lif multi-position XYZT stacks, but it should work with your data too.
Put all the XYZT files into a single directory with no other files, and the macro reads the number of files, slices, and positions, then does a bunch of automated cleaning including:
Normalizing the autofluor channel intensity to the GFP channel and subtracting from the GFP channel,
Applies a 1x1x1 median filter to reduce shot noise
Rescaling the contrast of each stack so the intensity remains constant and contrast is optimal, then downsampling to 8 bit
Removing any blank time points (sometimes lif files include a blank time point at the end if stopped early)
Adds or subtracts slices to make sure every stack has the same dimensions, then concatenates the timepoints.
Performs a 3D alignment of the stacks
Then after saving the ligned stacks it also creates a MIP of each stack for quick checking of the data.
The macro is also heavily commented, so you should be able to adjust it to your needs (see attached).
Hi Phillip,
A couple of suggestions:
1) if is a memory issue in ImageJ you can increase the memory under Edit>Options>Memory and Threads. I never used FiJi but I suspect there is a similar command.
2) Once you opened your files you can put them together in a stack using the command under Images>Stack>Images to stack. Then you can save it as TIFF stack or .avi.
3) There where a number of good updates in stack managing in the last years. I am not sure which version of ImageJ you have, but if haven't updated it in a while I would suggest suggest to do so.
4) Again, I am not sure on Fiji, but in ImageJ there are a number of plugins that allow you to open directly the .LSM files without the need to import as raw.
Hope this help, good luck!
You could use the Bio-Formats Importer plugin. In FIJI it is located under Plugins -> Bio-Formats -> Bio-Formats Importer.
Then you select one of the files. In the following dialog box you select 'Group files with similar names', 'Concatenate series when compatible' and 'Swap dimensions' (just in case FIJI swapped time and z). Also select 'Use Virtual Stack'.
After that it should ask you which pattern to use to determine the order of the files and then it should start loading/showing the image.
You simply open the file where you should go to z-project in order to make one single maximum projection file. Then the series of images can be converted from stacks to images.
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