Michael Hooker Microscopy Facility (MHMF.ORG)

Fixing X-Y Registration Shifts in Time Lapse Image Sequences

Introduction:

This section assumes that time lapse images have been gathered as .cxd format files using C-Imaging acquisition, and shows the steps to follow using the TurboReg plugin for ImageJ to reregister (realign) the images using rectilinear shifts.  Please note that these notes are terse!  Also more complex morphing is possible with the TurboReg plugin.  These brief notes about loading and viewing images with ImageJ may be helpful.  Note there is an overall intensity shift produced by TurboReg realignment, which can skew the result of intensity analysis.

• Get ImageJ and install it.
• Get TurboReg plugin and extract the files:
  • Put the extracted files into C:\Program Files\ImageJ\plugins\TurboReg (or equivalent)
  • From ImageJ run Plugins --> Short Cuts --> Install Plugin and select TurboReg_ from the Plugin: pull down menu
• Make copy of primary image data (good idea in case new SimplePCI version irreversibly changes the image files of version 5.3.1)
• Open .cxd image in SimplePCI
• Export Image Sequence:
  • File -->  Export Image Sequence --> All Components
  • 
  • Choose "Type" as Image Files
  • Choose Separate R, G, B files
  • 
  • Select Insert leading zeros
  • A series of Red & Green & Blue channel tiff files are generated, which will be imported in ImageJ for processing. 
• Run ImageJ
  • Import Red series
  • Import green series
  • Perform:
    • Plugins --> TurboReg --> TurboReg
    • Choose source (e.g. Red channel)
    • Choose target (e.g. green channel)
    • Select Translation
    • Choose land marks on each image
    • Hit Batch button
    • Covert image bit depth from 32 bit back to 16 bit (or 8 bit)
      • Image --> Type --> 16 bit
    • Save enhanced image series as .tif (multipage).
    • Repeat for Red series and save
    • Save enhanced image series as .tif (multipage)
• In SimplePCI
  • Import a RGB merged image series.
    • File -->  Import
    • 
    • Check Merge R, G, B files box
    • 
    • Choose browse and  select the multipage tiff file to import
    • Select again for additional channels, if any
    • Press OK
    • Check Add Field Details
    • 
    • Enter an appropriate time Incr., which must not be zero
    • Merge image should be made
    • Save the merged image
  • Inspect image series
  • Do analysis

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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