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Pre-processing

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Pre-processing of OpenSPIM data

The 11 multi-view time-points of a Drosophila embryogenesis time-lapse downloaded on the previous page are files saved directly from the OpenSPIM plugin during acquisition as OME Tiffs. Unfortunately, the current SPIM registration plugins are not able to open the files due to metadata conflicts.

Note: This is hopefully a temporary situation which will be remedied in the future and make the following pre-processing step obsolete. Please realize that Fiji, OpenSPIM and OME are large Open Source projects that need to communicate to make things like this work. Typically no-one is directly paid for this type of work and everyone is busy with what they are paid for doing. Nevertheless, it is our priority to make the handling of OpenSPIM data in Fiji as smooth as possible.

In order to use the OpenSPIM ome.tiff data in Fiji we need to save them as regular tiffs. We will take this as an opportunity to introduce the powerful ImageJ macro language that is very suitable to achieve such tasks with minimal programming experience. The following ImageJ macro was recorded in Fiji and edited in the Script Editor plugin.

for ( t = 0; t < 11; t++ )
{
	for ( a = 0; a <= 4; a++ )
	{
		open("/home/tomancak/Desktop/OpenSPIM_for_website/spim_TL" + IJ.pad(t, 2) +"_Angle" + a +".ome.tiff");
		saveAs("Tiff", "/home/tomancak/Desktop/OpenSPIM_for_website/tiffs/spim_TL" + IJ.pad(t, 2) +"_Angle" + a +".tif");
		close();
	}
}
Screenshot of Fiji Script Editor with re-saving ImageJ macro

The outer loop increments the time-points (we use built in ImageJ zero padding function IJ.pad(t, 2)), the inner loop increments the angle. The macro opens an ome.tiff in a directory /home/tomancak/Desktop/OpenSPIM_for_website, saves it in a sub-directory tiffs/ as .tif and closes the file.

Note: On a Windows machine the directory path would like this : C:\\home\\tomancak\\Desktop\\OpenSPIM_for_website\\

The easiest way to run the macro is to launch the Script Editor plugin (press letter l on the keyboard to launch Fiji Command Finder and then type Script Editor, then click on RUN). Paste the macro in the Script Editor. Select ImageJ macro from Language menu - this will result in helpful syntax highlighting. If necessary modify the numbers in the loops to reflect the input/output directory, the number of time-points (t) and angles (a) in your time series. Save the macro or simply run it.

It is a macro, therefore it will show every image it opens temporarily before saving and closing it. The downside of this is that the computer cannot be used for anything else during that operation.

Note: If you wish to run the plugin faster without showing the images, add this command as first line to the macro:

setBatchMode( true );

The end result of running the macro will be 55 new files (11 time-points x 5 angles) in the directory tiffs/. These are regular tif files that we can use for bead based registration in Fiji.