FRAP is based on the principal of observing the rate of recovery of fluorescence due to the movement of a fluorescent marker into an area (volume) of a cell or artificial construct which has same marker but which has been rendered non-fluorescent via intense photobleaching.  It can be useful for studying diffusion vs. motor driven recovery or lack of recovery.

PROTOCOL:

Startup:

1. Turn on system properly.
2. Choose method.
3. New Experiment. 
4. Set objective magnification
5. Set window size
6. Set levels for laser and gain and speed for scanning to get a pretty picture

For each experiment:

1. New Experiment
2. To get a pretty picture, set levels for laser and gain and speed for scanning and note the laser and zoom settings.
3. Center cell in field.
4. Do time lapse for a few timepoints to get a baseline.
5. Close image window.
6. New Experiment
7. Set up time series window so it is ready to go.
8. Zoom in to spot.  May have to type number into zoom value box.  Can go to 999.  
9. Crank laser to 100%
10. When bleached, laser to imaging intensity.
11. Zoom out  to imaging magnification.
12. stop laser scanning.
13. Time series.
14. close imaging window

Every Few Experiments:

In the window which lists the open experiments, click on an experiment and close it.   Close all or most experiments to free memeory and reduce chance of software crashing.  How to recover from a crash.

Shutdown:

See instructions at http://www.aecom.yu.edu/aif/instructions/BioRad_instructions/radiance.htm#shutdown

Analyzing data:

Use the measure spot through T sequence script in either NIH-Image or in IP Lab Spectrum.  This involves freehand drawing an ROI on the are to be measured.   This drawing can be done at any timepoint.  Do same area for each baseline T series and experimental T series

Save the lists of numbers.  The script is set for mean value, but you may add others such as area, max, min, stdev, etc.

Open in your favorite spreadsheet program.  It is advisable to subtract a background value from each timepoint from the FRAP spot for each timepoint.  Simply subtract across columns.

NIH-Image may make 0 white and 255 black.  In this case, make a column with the formula "=255-A1".

Page last revised 20 June 2001 by Michael Cammer 

Disclaimer:  images and graph are partial simulation.