ViBE-Z Data Preparation using ImageJ


Steps to reformat data from confocal stacks to the start of ViBE-Z

TESTED ON A WINDOWS 7 MACHINE -- other Windows versions and Linux should work as well

  1. Record stacks exactly as instructed by Rath et al. Nat. Protocols 2012 (doi:10.1038/protex.2012.031)
  2. Have ready (or similar other confocal multi-stack file)
  3. Have ImageJ/Fiji ready with PLUGINS
  4. Open dorsal stack in Bio-Formats importer with "Hyperstack" "autoscale" options (all other options off!)
    In ImageJ you should see a hyperstack window with channel, z and time dimensions If the order is not right, the HDF plugin will not save the proper formatted file for vibez. If order is not right, try to assemble in your Zeiss, Nikon etc software in proper order for initial export, or import stacks individually and build hyperstack in ImageJ in proper organization.
  5. Save dorsal hyperstack using File -- Save As... -- HDF5 (new or replace). Select output filename. IMPORTANT: Do not use spaces or other special characters in the filename. This will confuse ViBE-Z. A window pops up that lets you chose at top whether you save the dorsal or ventral stack. choose Presets: "LSM--> vibez step0 dorsal" and click [save]. This will automatically write all the stacks with proper dorsal stack information into the HDF5 format
  6. Open ventral stack in Bio-Formats importer with "Hyperstack" "autoscale" options see #4
  7. Saving: Choose File -- Save As... -- HDF5 (append) and the SAME FILENAME and directory as dorsal!) select Presets: "LSM--> vibez step0 ventral" and click on [Save] . This will automatically write all the stacks with ventral stack information into the HDF5 format The saved file should now be twice as large as the dorsal only version. You may test-open it in the HDF5 reader and will see there are 16 channels of the stack, with the proper annotations.
  8. (optional) Use the Vibez-Validate plugin by selecting Plugins -- HDF5 -- Vibez validate to check wether your created HDF5 file is correct, and if the data quality is sufficient.
  9. You should have a user id and password for the VIBE-Z server.
    Can get this from Olaf Ronneberger (ronneber@informatik.uni-freiburg.de).
    Upload HDF5 file by FTP onto main directory (FileZilla recommended!)
  10. On vibe-Z web interface, you will see the file in the your dataset list when upload is complete
  11. Check the file HDF5 file checkbox you want to process and start vibe-z.
    You can check progress by opening log file
    After about minimum time of 30 minutes the file is processed.
    Use FTP to download HDF5 file with same name as your upload to your local PC. The file may be 12 Gb large! So have some patience!
    The uploaded file contains original data, many intermediate quality controls saved, and the final registered channels 0 (reference stain) and 1 (signal)


Last Update: 31.8.2015