Author: Robert Bagnell (Bagnell@med.unc.edu) Pathology & Lab Med UNC-CH
History: 2011/7/01: First Version
Requires: ImageJ 1.44C or newer
Installation: Download Biosensor_FRET.ijm to the plugin folder and restart ImageJ. The macro also requires the following files to be installed in ImageJ:
- Biosensor_FRET_Names is a plugin that ask the user to assign the names of open stacks to names that Biosensor_FRET expects – available from MSL.
- StackAverage_16 is a16-bit version of the standard ImageJ plugin StackAverage - available from MSL.
- Infinity_to_zero is a plugin that sets any pixel in a 16-bit stack that is 65535 to 0 - available from MSL.
- Shading_Corrector plugin from the Official ImageJ plugins collection
- SubtractMeasuredBackground from the Official ImageJ macros collection
- TurboReg plugin from http://bigwww.epfl.ch/thevenaz/turboreg/
- DoRT_Curve_Fit_Bob is a plugin that utilizes the built-in ImageJ curve fitting routines to do a double exponential curve fit in the Photobleach correction section - available from MSL.
Description: This ImageJ macro processes time-lapse image stacks from live cells made using the single chain CFP-YFP FRET biosensor from Klaus Hahn for detection of RAC and RHO activity. The image processing steps are those outlined in the paper: Imaging Spatiotemporal Dynamics of Rac Activation in Vivo with FALIR by Hahn et al in Methods in Enzymology, Vol 325.
While the Hahn biosensor was the impetus for creating this macro, the macro can be utilized for any single chain biosensor exhibiting FRET, where it is not necessary to make bleed-through corrections. The macro is extensively documented in the source and is described in the MS Word document Biosensor_FRET Macro Description.docx available from Dr. Bagnell.
These files are provided for use under Open Software License v. 3.0 (OSL-3.0) Downloading or using any of these files constitutes your acceptance of the licensing agreement.