We typically generate a sequence of z series in which we accumulate 1 z series every minute or so. In order to then image the motility that is occurring in a particular image plane in the stack, it is necessary to track particular planes. Currently, we have a set of plugins that are modifications of plugins available on the ImageJ web site. They are in the Stacks to Movies zip file. Place the files in a folder named "Stacks to Movies" (or whatever you prefer) in the plugins folder of ImageJ and start ImageJ. Then under plugins, select the Stack to Movies option (if that is what you named the folder) and click on StackstoMovies. The plugin begins by asking for a name that will be attached to each movie slice, and then asks whether it is a blue green z series (files ending in 01.pic will be put in red and blue channels, files ending in 02.pic will be in green channels) or red/green z series (files ending in 01.pic will be put in green channel, files ending in 02.pic will be in red channel). These options can be modified by changing the js1stepRGB_Stack_Merge.java file. The program then loads the files, assembles color stacks, concatenates the stacks, rearranges them into movies of slices, and then checks where to save each one. The results can then be viewed using ImageJ and xy drift compensated using the plugins described in the next section.
Items to put in a file named "Stacks to Movies" in the Plugins folder:
Stacks to Movies folder files
Procedure
- Start ImageJ
- Select StackstoMovies from the plugin dropdown menu
- Provide a name to be appended to each slice number.
- Determine whether you want …01.pic files (option red) to be red or …02.pic files to be red (option 2).
- Select a place to store the completed slice movies and approve saving of each slice movie.
Possible memory issues for this step. It is necessary to have a machine with lots of RAM (probably twice the total amount in the z series files that you wish to convert to movies) because all the data are loaded into RAM in order to reshuffle them. In addition, an amount of RAM at least equal to the total file size must be allocated to ImageJ in the .cfg file before you run it (i.e., the -Xmx340m command could be changed to –xmx500m to correspond to 500 MB of RAM). If you have limited amounts of RAM you can combine just a fraction of your total z series, either reducing your time resolution, or else making movie fragments which you then can merge using various stack commands that can be downloaded from the ImageJ plugins download site.