The Fluorescence Recovery After Photo-bleaching (FRAP) experiment method consists of photo-bleaching a point (or points) of interest and then observing the recovery of fluorescence in the bleached area.
Open an image in the Image window. From the softWoRx main menu, choose Measure | PK Analysis. The Photokinetic Data Analysis window is displayed.
Tip You can use the Photokinetic Data Analysis window to specify a recovery model, type of ROI, beam profile shape, and number of sites. You can also use it to remove background intensity, select which Z sections and wavelengths to include in the analysis, and specify other options.
Drag the window number into the Input Image field.
Select the desired Response type, Recovery Model, and Data Recovery options.
If you are analyzing a multi-point FRAP data set, enter the number of laser sites in the Number of Laser Sites field.
If you are using background subtraction, enter a background value in the Background Intensity field.
To determine a number to enter in the Spot Radius field, use the Measure Distances tool to make an approximate measurement of the bleach spot.
Click Do It to run the analysis. The three channel output image file includes the original time lapse image, the ratio of the current time point to average pre-bleach time points, and the ratio data at the location used for analysis. A JPEG file contains a recovery graph that is a plot of the fluorescence intensity before and after the event. A log file contains the analysis results.