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
- Record stacks exactly as instructed by Rath et al. Nat. Protocols 2012 (doi:10.1038/protex.2012.031)
ready (or similar other confocal multi-stack file)
- lsm file with 8 stacks dorsal recording
- lsm file with 8 stacks ventral recording
Have ImageJ/Fiji ready with PLUGINS
- our HDF5 plugin (see HDF5 plugin webpage for installation)
- Bio-Formats plugin (this is part of the Fiji distribution)
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.
- 4 channels in the order:
- c1 stain signal low intensity
- c2 stain signal high intensity
- c3 nuclear stain low intensity
- c4 nuclear stain high intensity
- z planes as many as you have typically 400-500 (all substacks should have same number of images!)
- 2 "time points"
- t1 posterior part stack
- t2 anterior part stack
- 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
Open ventral stack in Bio-Formats importer with "Hyperstack" "autoscale" options see #4
- 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.
(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.
You should have a user id and password for the VIBE-Z server.
Can get this from Olaf Ronneberger (email@example.com).
Upload HDF5 file by FTP onto main directory (FileZilla recommended!)
On vibe-Z web interface, you will see the file in the your dataset list when upload is complete
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