Author: Robert Bagnell (email@example.com), UNCCH SOM Pathology & Lab Med MSL
History: January 2012: First Version
Requires: ImageJ 1.44C or newer
Installation: Download FRAP_Calculatorv3 to the ImageJ plugins or macros folder and restart ImageJ. Placing the macro in the plugins folder will cause the macro to appear in the plugins menu. The macro getStatusXYv5.ijm is required and must be in the ImageJ macros folder. Three other plugins are required if the camera dark noise and shading corrections are turned on: Stack_Average_16, FRAP_Calculator_Names, and Shading_ Corrector. These must also be in the ImageJ plugins folder.
Description: FRAP Calculatorv3.ijm macro calculates the Mobile Fraction, Immobile Fraction and Half Time for a fluorescence recovery after photobleach experiment. Prior to the calculation it corrects the data for photobleaching that is do solely to the imaging process and normalizes the photobleach curve to the first pre-bleach data point. In its default mode the mcaro does not make corrections for camera noise or shading. However, these corrections can be turned on by removing comment marks in the macro code. It is highly recommended that, if there is a free area of background that extends throughout the FRAP stack, a background correction should be done using the ImageJ macro Subtract_Measured_Background.txt.
The calculations are based on Rabut and Ellengerg in Chapter 7 of Live Cell Imaging A Laboratory Manual, Ed. Goldman and Spector, Cold Spring Harbor Laboratory Press 2005, ISBN 0-87969-683-4.
Data for the macro are a 12-bit or 16-bit time-lapse stack with some images prior to the photobleach and some images after the photobleach. The macro asks you to make a selection using an ImageJ drawing tool of the area that is photobleached. Then it creates a photobleach curve using the imageJ commands Plot Z-axis Profile. The macro then asks the user to select a second area for image corrections. This area could be the entire image if imaging a gel or a single cell if photobleaching a subcellular area. The macro creates a new graph based on this user-selected area. These data are then used to correct for photobleaching due to imaging the specimen and to normalize the photobleach data to the first time point to enable comparisons between experiments.
In the FRAP Calculator macro, the interval in milliseconds between images must be entered (100 is the default). The user is requested to click on the pre-bleach and stable post-recovery parts of the Corrected FRAP curve. Results appear in the Log window.
The macro requires the standard size plot window. You can check this in ImageJ by going to Edit/Options/Plot Profile Options and making sure width is set to 450 pixels and height to 200 pixels -this is the default plot size. The macro getStatusXYv5.ijm is required for interacting with the plot.
The FRAP Calculator macro can also make corrections for camera dark noise + shading (they are taken together). Shade + Noise can be turned on by removing the /*** and ***/ marks that surrounds that part of the code. (To view and edit the code, drop the FRAP_ Calculatorv3.ijm file on ImageJ.) They each require an additional image stack called “DARK” and “SHADE”. Both stacks should have the same dimensions in x, y and bit-depth as the FRAP stack. Both stacks should be made at the time the FRAP stack is made. DARK is a stack of images with no light going to the camera. SHADE is a stack of images, made using a uniformly fluorescent, un-bleachable specimen. Pixel intensity in SHADE should be in the 400 range on a 12-bit scale.
Shade + Dark requires the plug-ins Stack_Average_16 and Shading-Corrector.
As a convenience, the plug-in FRAP_Calculator_Names can be substituted for the opening "wait For User" command to input the file names for Shade and Dark stacks. Be sure to comment out the waitFor User and rename(“FRAP”) commands if you do this.
All of these macros and plug-ins are available at the web page www.med.unc.edu/microscopy in the zip file FRAP Calculator Package.zip.
Log table values:
Fpre = y value before photobleach
Fpost = y value after photobleach
Fend = y value where the recovery curve stabilizes
Tpost = time at which Fpost occurs
Thalf = time at which half the recovery occurs
Mobile Fraction = percentage of area below the curve
Immobile Fraction = percentage of area above the curve
HalfTime = amount of time required to reach half of full recovery
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.